Antibody library

ABSTRACT

An antibody library is prepared by selecting a light chain variable region capable of binding to the variable region of heavy chain to reproduce an active conformation and using the same. Because of being capable of maintaining the diversity of the heavy chain variable region at a high ratio in vitro, the antibody library of the present invention is expected as enabling the acquisition of antibodies with various binding activities.

TECHNICAL FIELD

The present invention relates to an antibody library comprising DNAsencoding antibody variable domains.

BACKGROUND ART

The animal body has the ability of producing antibodies specificallyrecognizing and binding to various structures (epitopes) on the surfacesof various foreign agents invading into body fluids. The size ofantibody repertoire (the total number of antibody types having distinctamino acid sequences binding to different types of antigens) of ananimal individual has been estimated to be approximately 1 to 100millions. The enormously large antibody repertoire is owing to DNArearrangements of heavy chain VH-D-JH and light chain VL-JL on theantibody locus during differentiation of bone marrow stem cells intoantibody-producing B lymphocytes. This event of DNA rearrangement occursindependently in each B cell. Thus, a single B cell that has a pair ofVH-D-JH and VL-JL genes produces only a single type of antibody.However, a collection of entire B cells in an individual can producevarious types of antibodies.

The techniques of antiserum preparation and monoclonal antibodypreparation by using cell fusion, both of which have been utilized inthe prior art, are based on the antibody-producing mechanism of animalbody. Specifically, an antigen substance is injected in combination withan adjuvant into an animal (rabbit, goat, mouse, etc.) several times ata certain intervals of time. When the animal immune system recognizesthe substance as a foreign one, a B cell that expresses an antibodybinding to the antigen substance is stimulated for growth anddifferentiation, and thus a large quantity of the antibody is initiatedto be secreted into the body fluid. Since there may be variousstructures on the surface of an antigen substance, even if it is apurified antigen, actually, the secreted antibody binding to the antigenis not a single type but a mixture of various types of antibodies. Aserum containing such an antibody mixture (antiserum) is called“polyclonal antibody”. Polyclonal antibodies have been used as usefulreagents for research. However, polyclonal antibodies which are reactiveto their target antigen substances are often cross-reactive to somemolecules having partly similar structures to the target antigenmolecules. Such cross reactivity has been problematic, when a polyclonalantibody is used as a reagent for detecting an antigen.

The cell fusion technology was established and changed the situationcompletely. There are many B lymphocytes producing antibodies binding toan antigen substance in the spleen in an animal immunized with theantigen. However, it is difficult to culture and keep such cells alivepermanently in vitro. Then, an idea was conceived thatantibody-producing cells, which proliferate permanently, could beestablished by preparing hybrid cells obtained by fusing cells of atumor cell line and antibody-producing cells; such a method wasestablished eventually. Since a fusion cell line (hybridoma) thusestablished is derived from a single antibody-producing cell and asingle tumor cell, the antibody produced by the cell consist of a singletype antibody; thus, the antibody is called “monoclonal antibody”. Thistechnology was established by Köhler and Milstein in 1975. A monoclonalantibody is a collection of homologous antibody molecules. Thus,monoclonal antibodies have been used as highly specific antibodies withless cross-reactivity. However, it has been pointed out that this methodhas the following problems that:

(1) it is required to prepare a large amount of purified sample ofantigen substance;

(2) the substance must be antigenic in an animal to be immunized withit; and

(3) a great expenditure of time and effort is required to establish amonoclonal antibody.

An enormous number of useful antibodies have been provided by using thetechnologies for producing polyclonal and monoclonal antibodies, givingproof of the usefulness of the technologies. However, it is also truethat there remain many difficult problems to be solved with respect tothese methods. For example, these methods cannot meet the demand ofpreparation of antibodies against various antigens in a short time or ofselectively preparing antibodies binding specifically to epitopes havingspecial structures. It has been awaited to establish a method forpreparing an antibody library comprising various antibody molecules,which ensures to isolate desired antibodies in a short time.Theoretically, the number of antibody types in such an antibody librarymust be comparable to the size of antibody repertoire in the animalbody. Actually, however, it is impossible to prepare such an enormouslibrary from animal cells. Monoclonal antibody preparation is nothingbut screening of a library of antibody-producing cells derived from theanimal body to obtain antibodies having desired reactivity. However, therepertoire in the library is greatly reduced during cell fusion or otherprocesses.

Then, a method has been proposed, which comprises an E. coli expressionsystem for antibody genes. Better et al., and Skerra and Plukthun havesucceeded for the first time in expressing antibodies havingantigen-binding activity in E. coli (Better, M., Chang, C. P., Robinson,R. R., Horwitz, A. H., Science 1988, 240:4855 1041-3; Skerra, A.,Plukthun, A., Science 1988, 240:4855 1038-41). They attached a sequenceserving as a secretory signal in E. coli to the N-terminus of antibody;thus, Fab-type and Fv-type antibodies were successfully produced andsecreted by E. coli.

Further, immediately after being established in 1988, the PCR technologywas utilized to amplify genes encoding antibody variable domains.Primers sequences to amplify all types of VHDJH and VLJL genes expressedin the animal body (particularly, human) were proposed (Orlandi, R. etal., Proc. Natl. Acad. Sci. USA. 1989, 86:10 3833-7; Sastry, L. et al.,Proc. Natl. Acad. Sci. USA. 1989, 86:15 5728-32). Then, vectors forproducing antibodies in E. coli were constructed by using antibody genesamplified with theses primers (Huse, W. D. et al., Science 1989,246:4935 1275-81; Ward, G. E. et al., J. Clin. Microbiol. 1989, 27:122717-23). At this stage, the repertoire size of antibody library wasgreatly increased. However, it was difficult to screen trace amounts ofantibodies produced in E. coli using their antigen-binding activities asindices. Efficient screening awaited the application of thephage-display method to antibody library preparation.

The phage-display method was devised by Smith in 1985 (Smith, G. P.,Science 1985, 228:4075 1315-7); the method comprises using filamentousbacteriophage, such as M13 phage, containing single-stranded circularDNA. The phage particle comprises cp8 protein that is a major protein ofthe phage particle, enveloping its DNA, and five molecules of cp3protein functioning at the time of phage infection to E. coli. In thephage-display system, a fusion gene is constructed to encode apolypeptide linked to the protein cp3 or cp8, and the fusion protein isexpressed on the surface of phage particle. Such a phage particlecarrying the protein with binding activity on the surface can beenriched based on the binding activity to its ligand. This method forenriching DNA of interest is called “panning”. Enriched phages containDNA encoding the protein having desired binding activity in theirparticles. The use of such filamentous phages as described above allowedthe establishment of a system where screening based on the bindingactivity and DNA cloning can be carried out with high efficiency(Published Japanese Translation of International Publication No. Hei5-508076). A method using a filamentous phage library has been reported,where antibodies can be expressed as Fab-type molecules (PublishedJapanese Translation of International Publication No. Hei 6-506836). Inthis report, the method comprises fusing the variable region with cp3 orthe like whose N-terminal portion has been deleted.

The phage-display system was used for producing antibodies; antibodyconsisting of the VH domain alone, or scFv-, Fv-, or Fab-type antibodywas expressed as a fusion with cp3 or cp8. The phage antibody binding toan antigen also comprises the antibody-encoding gene. However,antibodies, which were isolated from the antibody library at the verybeginning using the phage-display system, often had only lowerantigen-binding affinity. A method comprising artificially introducingmutations into genes was proposed to enhance the binding activity.Winter et al. provided an antibody library, from which high-affinityantibodies can be obtained, which contained antibodies having thesemi-artificial sequences which were prepared by inserting randomsequences between all pairs of VH or VL gene and JH or JL gene isolated(Nissim, A., Winter, G. et al., EMBO J. 1994, 13:3 692-8). De Kruif etal. also prepared an antibody library based on essentially the sameprinciple (de Kruif, J., Boel, E., Logtenberg, T., J. Mol. Biol. 1995,248:1 97-105). Vaughan et al. produced a sufficiently large antibodyrepertoire by expanding the library size (Vaughan, T. J. et al., Nat.Biotechnol. 1996, 14:3 309-14). Such strategies were indeed successfulwith respect to some limited types of antigens. However, even with suchstrategies, the probability of isolating desired antibodies stillremains unsatisfactorily low. For example, even with currently availabletechniques, it is impossible to construct a human antibody library fromwhich desired antibodies can be isolated with a probability comparableto that in the isolation of desired monoclonal antibodies using mice.Thus, a library consisting of more variations of antibodies is demanded.

An in vitro system faithfully mimicking the human's antibody-producingprocess is ideal to isolate antibodies binding specifically to variousantigens from an in vitro constructed library perfectly containing all,types of human antibodies. The antigen-binding moiety of an antibody islocated within the complementarity determining regions (hereinafterabbreviated as “CDR”), I, II, and III (six regions in total) of thevariable (V) domains at the N-terminal ends of both chains H and L. Thetotal number of amino acid sequence variations of the CDRs (includinglength variations), can be assumed to reflect the antibody repertoiresize.

With respect to the antibody repertoire, it is necessary to considerboth “naïve repertoire” before antigen invasion into the body and“antibody maturation” after antigen invasion. The active antibody geneencoding an antibody is created via DNA rearrangement. There are twoclasses of light chains: λ chain and κ chain; the gene encoding its Vdomain consists of VL gene and JL gene. There are 36 types of Vλ genesand 7 types of Jλ genes for the λ chain. During differentiation into Bcell, the VL-JL gene is created via DNA cleavage and rejoining in thevicinity of VL gene and JL gene of κ chain or λ chain for the lightchain. In most cases (two third), the segment of amino acids at 1st to96th is derived from the VL gene and another segment of amino acids at97th to 110th from the JL gene. However, after DNA cleavage, anexonuclease digests short portions of the DNA ends to be ligated, andthen V (D) J DNA recombinase (recombinases) rejoins the DNAs. This mayresult in differences of approximately ±3 amino acids in the size of theVL domain encoded by the VL-JL gene. In the light chain, CDR1corresponds to amino acids at 24th to 34th; CDR2, amino acids at 50th to56th; CDR3, amino acids at 89th to 97th. Thus, for the λ chain, thetotal number of variations due to their combinations can be calculatedby; (the number of Vλ genes)×(the number of Jλ genes)×(the total numberof gaps). However, the actual size of Vλ-Jλ gene repertoire is smallerthan 200 at the highest estimate. Because the Jλ genes carry similarsequences to one another, and 67% of gaps at the junctions are constantand the remaining 27% or more fall within ±1. The situation with regardto the κ gene is similar to that with λ gene. The total number of V κgenes is 37; the total number of Jκ genes is 4. Thus, the size of Vκ-Jκgene repertoire is also smaller than 200.

The diversity in the light chain variable region is relatively low, butthe diversity in the heavy chain variable region considerably larger.CDR1 (amino acids at 31st to 35th) and CDR2 (amino acids at 50th to65th) are encoded by any one of 36 types of VH genes, and consequentlythe variety in this region is not so large. However, CDR3 producesenormous variations. CDR3 is positioned between CDR1 and CDR 2 of thetwo chains H and L in the antigen-binding moiety of an antibody. Heavychain CDR1, CDR 2, and CDR 3 comprise about 60% and the light chaincomprises about 40% of the whole surface area of the antigen-bindingmoiety. With respect to the portion excluding heavy chain CDR3, therepertoire size is estimated by: the number of light chain variations(several hundreds at the maximal estimate)×the number of heavy chainCDR1 and 2 variations (36)=approx. 10,000. Heavy chain CDR3 is encodedby a separate gene, which is called “D gene”; there are 26 types of Dgene variations. Two types of DNA rearranging events, namely D-JHrecombination and VH-D recombination, produce VH-D-JH and thus theCDR3-encoding region is completed.

It should be noted that the DNA rearrangement comprises the followingprocesses:

(1) DNA cleavage at positions immediately adjacent to the signalsequences in the vicinity of VH, D, and JH genes;

(2) Digestion of DNA at its terminal portions by exonuclease;

(3) Insertion of a random sequence (referred to as N) by terminaltransferase; and

(4) DNA repair and ligation.

In the above-mentioned process (2), larger variations are produced forthe heavy chain than for the light chain. Further, the presence ofprocess (3) is a more notable difference; light chain rearrangement hasno such process. Heavy chain CDR3 (corresponding to amino acids at 95thto 102nd) is a region located between cysteine at residue 92nd andtryptophan at residue 103rd. The length of the region is altered rangingfrom 5 to 20 amino acids or more, and the sequence is also highlydiverse in the region. These specific features produce an enormousnumber of variations of CDR3, the sequence of which, in effect, isdifferent in every B cell differentiated independently.

The DNA rearrangements of heavy chain VH-D-JH and light chain VL-JL inthe antibody locus during B cell differentiation is independent of thepresence of antigen. An entire population of antibodies produced bytotal B cells, each of which expresses a pair of VH-D-JH and VL-JLgenes, is referred to as “naïve repertoire of antibody”. After antigeninvasion, cells expressing antibodies capable of binding to antigens arestimulated for growth and differentiation. While secreting antibodies, Bcells are subjected to mutations frequently in the variable region gene(VH-D-JH, VL-JL) encoding the antibody. B cells producing antibodieswhose binding affinity for antigens is increased by the introducedmutations survive as memory cells while secreting the antibodies of highperformance. This process is referred to as “antibody maturation”. Themutational event plays the most important role in this process. Anyantigen specificity that is not originally present in the naïve antibodyrepertoire is never newly generated through the introduction ofmutations. Accordingly, a mechanism, by which clones having antigenspecificities that are absent in the naive repertoire should beeliminated, is required to construct an antibody library in vitro whichmimics the in vivo process of antibody production.

Problems on preparing antibody libraries in vitro are listed below.

(1) After each antibody gene is expressed, for example, in E. coli on alarge scale, the heavy chain and light chain variable domains of thegene products hold together to an antibody molecule though proteinfolding. Clones, whose gene products fail any of these processes andthus are incapable of forming the exact immunoglobulin conformation, areof no use.

(2) In vivo, a complex formed from a pair of heavy chain variable domainand light chain variable domain exhibits unique antigen specificity ineach cell; in vitro, it is necessary to construct a library by combiningseparately prepared libraries for the populations of heavy chainvariable domains and light chain variable domain. For example, when a Bcell population contains 10,000 types of cells, theoretically, theentire variations can be covered by a library at least consisting of:(10,000 types of the heavy chain variable domains)×(10,000 types of thelight chain variable domains)=100 millions in total. As the number ofcombinations is increased, the library size indeed becomes larger, butthe percentage of clones of inactive antibodies is also increased in thelibrary.

(3) When human blood is used as an antibody gene source, the expressionprofile of antibody genes of each person may have a significant biasdepending on his/her immunological history.

All of the above-listed three problems are involved in the causes ofbiased library repertoire. Namely, these result in unfavorable gapsbetween the theoretical and actual numbers of cloned in a library: thenumber of types of functional phage antibodies in a library preparedactually is markedly reduced as compared with the number oftheoretically estimated clones in such a library.

More specifically, a library may comprise clones whose distribution ishighly biased when the immune response against a specific antigen isenhanced. Alternatively, an antibody library prepared may contain manyclones encoding antibodies having only insufficient antigen-bindingactivity. For example, when 50% each of light chain variable regiongenes and heavy chain variable region genes encode active antibodymolecules, the probability that a combination of the two domainsproduces an active antibody is only 25%.

Such libraries have many problems in addition to one that the actualrepertoire size is far smaller than the theoretical size. For example,antibody molecules having only weak antigen-binding activity interferewith immunological reaction on screening. Specifically, antibody-antigencomplex formation is an equilibrium reaction; when clones of minoritypopulation coexist with those of majority population, the majority mayoverwhelm the minority in spite of the difference in the antigen-bindingaffinity.

In addition, the presence of clones encoding inactive antibodies can bean obstacle in cloning. Namely, as the number of clones encodinginactive antibodies is larger, the probability that the populationinclude clones proliferating very rapidly becomes greater. Such clonesgrowing rapidly are preferentially selected during screening, and thusmay cause a considerably high background.

A problem of previously reported libraries is that it is hard toestimate how many effective clones are actually present in thelibraries. It is thus impossible to evaluate the efficiencies of libraryand screening.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to provide an antibody librarycontaining antibody molecules having functionally active conformation ata high rate. Another objective of the present invention is to providesuch an antibody library, a method for producing the antibody library,and screening method using the antibody library. Still another objectiveof the present invention is to provide a method for isolating genesencoding immunoglobulin light chains ensuring antibody molecules havingthe functional active conformations.

The present inventors strenuously studied on factors obstructive toscreening a known antibody repertoire for desired antibodies. Then, theyconceived that the abundance ratio of antibody molecules havingfunctionally active conformation in the repertoire is too small toisolate useful antibodies from antibody libraries known to those skilledin the art. Various ideas have been proposed to establish previousmethods for producing antibody libraries known to those skilled in theart; the goal is to construct antibody libraries in vitro faithfullymimicking the in vivo antibody repertoire.

In some instances, methods even comprise introducing artificialmutations at random to increase the number of antibody variations.However, in most of such attempts, antibody molecules havingfunctionally active conformation were indeed produced, but antibodymolecules having only insufficient activity were also produced. Thus, anantibody library constructed according to any one of previous method hasan antibody repertoire containing antibodies of no use in vivo and manyinactive antibody molecules, which can result in unsuccessful screeningfor functionally active antibodies. For example, it is assumed that alibrary is prepared by combining independent 10⁵ clones for the lightchain variable region gene with 10⁹ clones for the heavy chain variableregion gene. The number of variations of heavy chain variable regiongene is sufficiently large. However, when there are many inactive genesin the variations given for the light chain variable region gene, a partof variations for the heavy chain variable region gene are eliminatedduring screening because their products cannot acquire the activity asthe immunoglobulin.

The living body has the mechanism of selective growth of clonesproducing antibodies having high reactivity as well as the mechanism ofproducing various types of antibody molecules at random. Not only thestep of eliminating inactive antibodies but also the step of increasingantibody variations is required to precisely mimic the in vivo antibodyrepertoire in vitro.

In this background, the present inventors predicted that antibodyisolation could be achieved more efficiently by increasing the abundanceratio of functional antibody molecules in the antibody library. To meetthis goal, it is necessary to identify the cause of the generation ofinactive antibody molecules in antibody libraries known to those skilledin the art. The present inventors focused on the role of the light chainin the maintenance of antibody activity. First, they established amethod for screening for the light chain allowing the formation offunctionally active conformation in an antibody molecule.

Further, the present inventors carefully analyzed the structures of theselected light chain variable region genes by the above-mentionedmethod, and then found that the use of only light chain variable regiongenes having limited structures enabled to construct an antibody librarycontaining antibody molecules having functionally active conformation ata high rate; thus the present invention was completed. There may be thepossibility that the size of antibody repertoire is reduced during thestep of selecting light, chain variable region genes. However, thepresent inventors carefully analyzed the structures of the light chainvariable region genes selected according to the selection method of thepresent invention, and then found that the number of structures of thelight chains capable of forming functionally active conformation whencombined with the heavy chains fell within a limited range. In thepresent invention, it was clarified that such structures of the lightchains constituting functional antibody molecules could be includedwithin a particular range of repertoire; the most striking feature ofthe present invention is thus the use of these light chain genes inlibrary preparation. Namely, the present invention relates to a method,which comprises the steps described below, for preparing animmunoglobulin gene library, rgdp library based on this library, and amethod for screening these libraries for genes encoding antibodiesrecognizing specific antigens. The present invention also provides amethod, which comprises the steps described below, for selecting genesencoding light chains capable of re-holding functionally activeconformation with the heavy chains.

[1] A method for preparing a gene library comprising combinations oflight chain variable region genes and heavy chain variable region genesof immunoglobulin, the method comprising:

(a) selecting light chain variable region genes encoding light chainmolecules capable of re-holding functionally active conformation with anexpression product of the heavy chain variable region genes;

(b) constructing a gene library which is a collection of the light chainvariable region genes obtainable in step (a); and

(c) combining the library obtainable in step (b) with a library of genesencoding the heavy chain variable regions.

[2] The method according to [1], wherein libraries of heavy chainvariable region genes which have been prepared separately for each VHfamily in step (c) are combined together in accordance with the in vivoratio of the respective VH families.

[3] The method according to [1], wherein the gene libraries in step (c)are combined in a single vector.

[4] The method according to [1], wherein the method further comprisesthe following step:

(d) selecting clones expressing the heavy chains using, as an index, alabeled peptide fused with the heavy chain variable regions.

[5] A gene library that is obtainable by the method according to [1].

[6] A gene library comprising at least genes encoding light chainvariable regions of immunoglobulin, wherein genes encoding light chainvariable regions incapable of re-holding functionally activeconformations with immunoglobulin heavy chain variable regions have beensubstantially eliminated from the gene library.

[7] The library according to [6], wherein the library has been combinedwith a library of genes encoding the heavy chain variable region.

[8] The library according to [7], wherein each VH-family gene libraryfor heavy chain variable region contains clones enough to cover in vivodiversity.

[9] The library according to [6], wherein the genes constituting thelibrary have been introduced into a host cell selected from the groupconsisting of bacterium, yeast, and plant cell.

[10] The library according to [6], wherein the genes constituting thelibrary have been introduced into mammalian cells.

[11] The library according to [6], wherein at least a part of the genesconstituting the library has been introduced into filamentous phages.

[12] The library according to [11], wherein fragments of the heavy chainvariable region and light chain variable region encoded by the genesconstituting the library are displayed on the surface of filamentousphages and re-hold a functionally active conformation.

[13] The library according to [12], wherein a gene encoding a labeledpeptide has been fused with the heavy chain variable region.

[14] The library according to [6], wherein the immunoglobulin lightchain variable region gene is derived from human.

[15] The library according to [14], wherein an immunoglobulin lightchain hypervariable region comprises an amino acid sequence withoutcysteine residue.

[16] A rgdp library, wherein each clone constituting the gene libraryaccording to [5] includes an antibody protein encoded by the gene in theclone.

[17] An antibody library that comprises antibody proteins encoded by thegenes in the respective clones constituting the gene library accordingto [5].

[18] A method for selecting genes encoding light chain variable regioncapable of re-holding functionally active conformation with the heavychain variable region, the method comprising:

(a) obtaining one or more genes encoding the light chain variableregion;

(b) obtaining a gene encoding the immunoglobulin heavy chain variableregion, wherein it is confirmed that the heavy chain variable regionre-holds functionally active conformation with the light chain variableregion;

(c) selecting arbitrary one of the genes encoding light chain variableregions obtained in step (a), and translating the selected gene into aprotein under a condition ensuring the re-holding of functionally activeconformation of immunoglobulin with the heavy chain variable regionencoded by the gene which has been obtained in step (b);

(d) detecting the formation of an antigen-binding moiety in the proteintranslated in step (c); and

(e) selecting a gene encoding the light chain variable regionconstituting the protein in which the formation of an antigen-bindingmoiety has been detected.

[19] The method according to [18], wherein the method further comprisesthe following step:

(f) determining the nucleotide sequences of the light chain variableregion genes, comparing the amino acid sequences encoded by thenucleotide sequences with each other, and eliminating genes encodingidentical amino acid sequences and genes encoding deleted amino acidsequences.

[20] The method according to [18], which comprises expressing the lightchain variable region of immunoglobulin on the surface of filamentousphage.

[21] The method according to [18], which comprises using, as a samplecontaining the light chain to be used in step (c), the culturesupernatant of a host microorganism infected with phagemid into which agene encoding the light chain variable region of immunoglobulin and agene encoding the heavy chain variable region have been inserted.

[22] A method for detecting an immunoglobulin variable region that bindsto a particular antigen, the method comprising:

(a) contacting the antigen with the library according to [5] or [7] orwith an expression product of the library under a suitable condition forantigen-antibody reaction; and

(b) detecting the binding between the antigen and the immunoglobulinvariable region.

[23] The method according to [22], wherein the library is the libraryaccording to [12].

[24] A method for isolating a immunoglobulin variable region that bindsto a particular antigen, the method further comprising carrying out thefollowing step after practicing the method according to [22]:

(c) selecting a clone expressing the immunoglobulin variable regionwhich binds to the above-mentioned antigen.

[25] The method according to [24], wherein the method further comprisesthe following steps:

(d) preparing a secondary library by amplifying phage clones selected instep (c); and

(e) repeating steps (a) to (d) for the secondary library until therecovery rate of clones selected in step (c) is elevated.

[26] A clone or immunoglobulin fragment isolated by the method accordingto [24], or a gene encoding the same.

[27] A polynucleotide comprising any one of the nucleotide sequences ofSEQ ID NOs: 61 to 78.

[28] A protein comprising any one of the amino acid sequences of SEQ IDNOs: 79 to 96.

[29] A kit for preparing an antibody library, the kit comprising thefollowing:

(a) a light chain variable region gene library from which genes encodinglight chain variable regions incapable of re-holding functionally activeconformation with heavy chain variable regions have been substantiallyeliminated; and

(b) a set of primers with which the genes encoding the heavy chainvariable region can be amplified.

[30] A method for preparing an antibody library, the method comprising:

(a) introducing a gene encoding a region containing at least a variableregion of immunoglobulin into phagemid;

(b) infecting a host microorganism with the phagemid obtained in step(a); and

(c) recovering, as an antibody library, the culture supernatant of thehost microorganism of step (b) without infecting helper phage.

In addition, the present invention relates to a method for preparingantibodies recognizing the pathogens described below.

[31] The method according to [24], wherein the antigen is an antigenderived from a pathogen.

[32] A method for preparing an antibody having activity of neutralizinga pathogen, the method further comprising carrying out the followingstep (f) after practicing the method according to [24]:

(f) evaluating an antibody variable region selected in step (c) for theactivity of neutralizing the pathogen and selecting an antibody variableregion having neutralizing activity.

[33] The method according to [31], wherein the antigen derived from apathogen is an antigen selected from the group consisting of influenzavirus HA antigen, diphtheria toxin, tetanus toxin, and varicellavirus-derived glycoprotein.

[34] The method according to [31], wherein the following step (a′) ispracticed before step (a):

(a′) contacting the library with an antigen for absorption and removingantibodies bound to the antigen for absorption from the library;

wherein the antigen for absorption refers to an antigen

-   -   which derived from the same pathogen from which the        above-mentioned antigen is derived and    -   antibodies reactive to which is undesired to be isolated.

[35] Use of the library according to [12] for preparing a neutralizingantibody against a pathogen.

As used herein, “immunoglobulin” refers to every type of immunoglobulinmolecule consisting of heavy chain and light chain regardless of typesof antibody class and animal species. The “immunoglobulin” also includesa fragment consisting of a domain capable of binding to an antigen and achimeric antibody which is composed of multiple immunoglobulin domainsderived from two or more animal species. In general, mammalian genesencoding the heavy chain variable region have been categorized intoseveral VH families based on the structural features of the gene. Forexample, the human genes have been categorized into 7 families of VH1 toVH7. Members of the respective families contain nucleotide sequenceshighly conserved between the families. Based on the highly conservedsequences, PCR primers have been designed to amplify the members of eachfamily. Like the heavy chains, the light chain variable domains can becategorized into several families based on the structural features.

A gene encoding the heavy chain variable region is consists of threeclasses of genes, namely V (variable), D (diversity), and J (junction).Each gene class V, D, or J comprises multiple genes; random combinationsof these genes and introduction of mutations result in the antibodydiversity. On the other hand, the light chain variable region consistsof two classes of genes V and J. Combinations of multiple gene classesand introduction of mutations results in the diversity of the lightchain variable region as well as the heavy chain variable region.

The term “library” is used herein, which refers to a collectioncomprising a repertoire of various components. Gene library, antibodylibrary, and phage library are composed of genes, antibody molecules,and phages or phagemids, respectively. When an antibody gene in a phagegenome is expressed on the surface of phage particle, such a genelibrary is an antibody library. However, as used herein, the term “phagelibrary” also refers to a library of phage on which antibody moleculesare not expressed. Specifically, cells of host microorganism infectedwith phagemid and lysogenized phages containing a genome carrying genesencoding the antibody variable region are also referred to as “phagelibrary”.

Further, the term “rgdp library” (replicable genetic display packagelibrary) is used herein. The rgdp library refers to a library comprisinggenes and the expression products of the genes displayed on the surface.When members of the above-mentioned phage library express antibodyproteins on the surface of phage particles, the library is an rgdplibrary. Such rgdp libraries include a library comprising transformedcells expressing foreign proteins on their surface or ribosomes inaddition to the phage library.

Further, the term “conformation” is used herein. As described above,immunoglobulin is a complex formed by holding of heavy chain and lightchain. The conformation refers to the resulting structure of the complexthrough the heavy chain-light chain association. Basically, theconformation is established by disulfide bonding in the constant region.However, such an immunoglobulin does not always acquire antigen-bindingactivity. In the present invention, when a certain immunoglobulin hasantigen-binding activity, one can state that the conformation of theimmunoglobulin is functionally active. When a heavy chain which forms afunctionally active conformation in combination with a certain lightchain forms a functionally active conformation in combination withanother light chain, then the association of the two is particularlyreferred to as “re-holding”. Another light chain which constitutesre-holding includes a light chain isolated as a separate clone from anidentical cell. Further, as used herein, “re-holding of conformation”basically refers to the re-holding of a region required for the bindingof immunoglobulin to an antigen. Thus, it is assumed that thefunctionally active conformation is re-holded when the molecularstructure in the variable region is re-holded as an immunoglobulinmolecule regardless of the presence of constant region. Further, it isassumed herein that the functionally active conformation is formed whenthe re-holding is achieved in the variable region, even if artificialnucleotide sequences have been inserted in the genes encoding the lightchain and heavy chain or even when genes encoding phage proteins havebeen fused with the immunoglobulin genes. More specifically, as usedherein, the term “re-holding of molecular structure” equivalently meansthat the heavy chain variable domain and light chain variable domainconstitute the immunoglobulin variable domain via disulfide bonding inthe constant region, for example, when the genes encoding the heavychain variable region and light chain variable region are translated toseparate proteins.

On the other hand, there are antibody molecules in each of which theheavy chain and light chain have originally been linked together via anartificial linker, such as single chain Fv antibody (scFv). In suchspecial types of antibodies, their conformations are established via notdisulfide bonds but peptide bonds in some cases. Thus, the conformationof scFv antibody can be re-holded not through the constant region.

Firstly, the present invention relates to a method for preparing a genelibrary, which comprises selecting genes encoding light chains capableof re-holding functionally active immunoglobulin molecules and combiningsuch a light chain variable region gene with a library of genes encodingheavy chains. The selection of light chain variable region genes of thepresent invention can be achieved by the steps described below.Specifically, the present invention relates to a method for selectinggenes encoding light chains that allows re-holding functionally activeconformation when combined with a heavy chain, which comprises thefollowing steps:

(a) obtaining one or more genes encoding the light chain variableregion;

(b) obtaining a gene encoding the immunoglobulin heavy chain variableregion that has been confirmed to re-hold a functionally activeconformation when combined with the light chain variable domain;

(c) selecting arbitrary one of the genes encoding the light chainvariable region obtained in step (a), and translating the same into theprotein under a condition ensuring the re-holding of functionally activeconformation of immunoglobulin, when combined with the gene encoding theheavy chain variable region which has been obtained in step (b);

(d) detecting the formation of antigen-binding moiety in the proteintranslated in step (c); and

(e) selecting a gene encoding the light chain variable domainconstituting the protein whose antigen-binding moiety has been detectedto be formed.

In the present invention, the light chain variable region or heavy chainvariable region may be an arbitrary region comprising at least a portionrequired for antigen binding. In other words, an arbitrary regioncontaining a region comprising three CDRs and the frame (FR) containingthe same can be used as a variable region of the present invention.Accordingly, for example, a fragment containing the constant region canalso be used as a variable region of the present invention, as far as itcontains the region required for antigen-binding. Fab and Fab′, whichare often used as antibody variable domains, are names originallyprovided for fragments obtained by enzymatic digestion ofimmunoglobulin. As used herein, “Fab” is construed not to be a term tolimitedly specify the variable region.

A light chain variable region gene, which is a target in the method ofthe present invention for selecting light chain variable region genes,can be obtained from an arbitrary antibody-producing cell. Suchantibody-producing cells include, for example, peripheral bloodlymphocyte and splenocyte. RT-PCR can be used advantageously to isolatelight chain variable region genes. For example, primers that can be usedfor amplifying the human VLJL gene, have been disclosed (PublishedJapanese Translation of International Publication No. Hei 3-502801; orPublished Japanese Translation of International Publication No. Hei4-500607). In addition, such primers are also publicized on the homepageof MRC Corporation (“V-base”). Genes encoding the light chain variableregion to be used in step (a) can thus be obtained by PCR using theseprimers. The genes obtained are used in step (c).

Then, genes encoding immunoglobulin heavy chain variable domains thathave been confirmed to allow the re-holding of, functionally activeconformation when combined with a light chain variable domain areisolated in step (b). The heavy chain variable region to be isolated inthis step may have arbitrary antigen-binding specificity or the like, asfar as it is derived from the same animal species from which the lightchain variable region is obtained in step (a) and allows the re-holdingof functionally active conformation in combination with the light chainvariable region. A gene encoding such a heavy chain variable domain canbe obtained, for example, from a gene encoding an immunoglobulinmolecule that has been demonstrated to exhibit the activity as anantibody. It is preferable that a heavy chain variable domain to be usedin step (b) should be prepared as one that can re-hold with κ chain or λchain. It is preferred to select a heavy chain variable region with thehighest efficiency by practically testing the efficiency of holding withthe light chain. For example, in Example described below, when variousclones for the heavy chain were evaluated for the efficiency of holdingwith the light chain, then VH3-4 having the following primary structureshowed the highest efficiency of holding; VH3-4 (SEQ ID NO: 1) was thusselected, and has the following primary structure.

FR1: EVQLVESGGGLVQPGRSLRLSCAASGFTFD

CDR1: DYAMH

FR2: WVRQAPGKGLEWVS

CDR2: GISWNSGSIGYADSVKG

FR3: RFTISRDNAKNSLYLQMNSLRAEDTALYYCAK

CDR3: GPSGSFDAFDI

FR4: WGQGTTVTVSS

Then, arbitrary one of the genes encoding the light chain variableregion obtained in step (a) and the gene encoding the heavy chainvariable region obtained in step (b) are both translated into proteinsunder a condition ensuring the re-holding of functionally activeimmunoglobulin conformation in step (c). Since genes encoding the heavychain variable domains capable of re-holding a functionally activeconformation have been selected in step (b), all the molecules allowingthe re-holding of the conformation for the variable region ofimmunoglobulin molecule in step of (c) can be assumed to havefunctionally active conformations. As far as the above-motionedimmunoglobulin molecule contains the essential portion for the antigenbinding of immunoglobulin, any type of molecular organization isallowable for the above-motioned immunoglobulin molecule. Thus,regardless of the presence of constant domain, the molecule can beassumed to be a re-holded immunoglobulin molecule when having there-holded antigen-binding moiety.

The “condition ensuring the re-holding of immunoglobulin” in step (c)refers to a condition under which disulfide bonds enable holding betweenthe heavy chain variable domain and light chain variable domain. Morespecifically, for example, the in vivo reducing environment (e.g., in E.coli periplasm) as described above with respect to the expression of Fabprotein is a condition ensuring the re-holding of immunoglobulin. Areducing microenvironment required for the acquisition of the antibodyconformation can also be provided by organelle such as endoplasmicreticulum in cells derived from mammals including human. Further, insome cases, such reducing environments are not required for re-holdingimmunoglobulin, when an antibody consists of the heavy chain and lightchain variable domains linked together via an artificial amino acidsequence (linker), e.g., an scFv-type antibody.

Phages that express foreign genes on the surface can be usedadvantageously to express the light chain variable domain and heavychain variable domain in step (c). For example, filamentous phages canexpress a fusion protein on the surface, which comprises a proteinencoded by a foreign gene and a phage protein such as cp3 or cp8.

Typical phage library screening comprises the step of recovering phageparticles. Thus, for example, when phagemid into which a foreign genehas been inserted, is infected, phage particles can be recovered byinfecting helper phage. On the other hand, the present inventors havefound that when E. coli infected with a phagemid containing, as aninsert, the Fab gene, which is fused with the cp3 gene, is culturedwithout adding helper phage, the fusion protein of Fab and cp3 issecreted into the culture supernatant. Only a trace amount of the fusionprotein of Fab and cp3 secreted from the E. coli infected with thephagemid was detected even after 20-hour culture, but it was enough forpracticing the selection method for the light chain according to thepresent invention. Thus, samples to be used in the method for selectingfor the light chain variable region gene according to the presentinvention may include culture supernatants of host microorganismsinfected with such a phagemid. This method which does not require thestep of recovering phage particles by infecting helper phage comprisesonly a very simple experimental procedure. A vector containing anoperative promoter in a host microorganism and a signal sequence is usedto prepare, as screening samples, culture supernatants of a hostmicroorganism infected with the phagemid according to the method of thepresent invention. For example, a phagemid vector of filamentous phage,into which the pelB sequence or the like has been inserted as a signalsequence, can be used, when E. coli is used as the host.

The immunoglobulin variable region is structurally formed, when a lightchain variable domain allows the re-holding of the functionally activeconformation in combination with a heavy chain variable domain. The typeof light chain variable region to be selected can be identified throughdetecting such structural formation of the variable domain. Theformation of variable domain can be detected, for example, by using theprinciple of immunoassay. Specifically, the light chain variable domainis trapped on a plate, on which an antibody against the κ chain (or λchain) has been immobilized, by adding a sample containing expressionproducts of the heavy chain variable region gene and light chainvariable region gene to the plate coated with the antibody. When theheavy chain variable domain is associated with the light chain variabledomain, the heavy chain variable domain, along with the light chainvariable domain, must be trapped on a plate. A labeled antibody againstthe heavy chain or Fab is then added to the plate. Only when the twotypes of molecules are associated together, the labeled antibody istrapped on the plate. After incubation for a convenient period, theplate is washed; a light chain variable domain re-holding thefunctionally active conformation can be identified by detecting thelabeled antibody. The labeled antibody and the immobilized antibody canalso be used in the inverse combination. Alternatively, it is possiblethat the heavy chain variable domain is pre-biotinylated, and then thedetection is carried out using labeled avidin. As described above, theinventors have revealed that culture supernatants of E. coli cellsinfected with phagemid can be used as samples in this detection method.

Such a light chain variable domain that has been confirmed to beassociated with the heavy chain variable domain by the method asdescribed above can be selected as a light chain variable domain thatallows the re-holding of the functionally active conformation incombination with the heavy chain variable domain. When a phage librarycontains genes encoding the light chain variable region, genes of thelight chain variable region can be selected by recovering the phageparticles.

Such light chain variable region genes obtained by the steps asdescribed above not only allow the re-holding with the heavy chainvariable domain but also are demonstrated to be expressed in theexpression system used in the screening. For example, when a phageexpression system is used, light chain variable region genes whoseexpression levels in E. coli cells are high enough are selected. Thus,genes whose expression levels in E. coli cells are too low can beeliminated, even if they are expressed in mammalian cells. Thus, themethod of the present invention for selecting light chain variableregion genes has such a new merit. On the contrary, with theconventional techniques for preparing antibody libraries, the lightchains are selected without such a selection step, and therefore it isimpossible to avoid the contamination of light chain genes whoseexpression levels are insufficiently lower.

Selected genes encoding the light chain variable region can be usedwithout any modification for preparing gene libraries of the presentinvention. However, at this stage, the selected genes encoding the lightchain variable region may have redundancy. Thus, it is preferred toremove redundant genes through analyzing the structures of the lightchain variable region genes before preparing the gene library usingthem. Such redundant genes can be removed, for example, by the followingmethod.

First, before or after the above-mentioned step (d), the nucleotidesequences of the light chain variable region genes are determined andthe amino acid sequences encoded by the nucleotide sequences arededuced. The deduced amino acid sequences are compared with one another,and then genes encoding identical amino acid sequences are removed. Itis preferred to additionally carry out deletion check at this stage. Forthis purpose, genes having reading frame shifts are removed after thenucleotide sequences are determined.

In practice, the selection and isolation of genes can be carried out bygrouping genes showing similarity into a single category, and selectinga representative sequence from each group. The selection should becarried out so as to cover all the selected genes without bias and notto alter the distribution of gaps at the VL-JL junction in thepopulation of naturally occurring antibodies. In practice, light chainvariable region genes for known antibody molecules were selected from agene database, and then the distribution was determined based on theresult obtained by analyzing gaps at the junction. Table 1 shows aresult of analysis for the number of amino acids at the V-J junction(the amino acid sequence 91-96).

TABLE 1 Group Size 1 2 3 4 5 Total Reported 1 0 0 0 0 0 0 1 (0.2%) 2 0 01 0 0 1 (1.0%) 2 (0.3%) 3 0 0 0 0 0 0 2 (0.3%) 4 0 0 0 0 0 0 1 (0.2%) 52 1 3 1 1 8 (7.9%) 54 (8.5%) 6 24 17 15 9 4 69 (68.3%) 427 (67.5%) 7 3 113 1 1 18 (17.8%) 119 (18.8%) 8 2 1 1 0 0 0 1 (0.2%) 11 0 0 1 0 0 1(1.0%) 0 Total 31 20 33 11 5 101 633 Group: Vκ family No. Size: thenumber of amino acids in the region of amino acid sequence 91-96

After general consideration of the results obtained as described above,according to the analysis result by the present inventors, therepertoire size of representative light chain variable region sequences,which were selected by the selection method, is 101 for the κ chain, orsimilarly 99 for the λ chain in the case of human immunoglobulin.

Thus, the repertoire size of the representative light chain variableregion sequences of functional human immunoglobulin was revealed to be200 at most. However, the repertoire size is not limited to the estimateof about 100 by the present inventors. Specifically, for example, whenone intends to select human light chain variable region genes accordingto the selection method of the present invention, the number of lightchain variable region genes to be selected is not always 100. The mostimportant thing is that light chain genes selected by practicing theselection method described herein based on the obtained amino acidsequences are used in the subsequent steps.

The VL genes of phage antibodies obtained by screening were categorizedherein into several groups. The result is shown in FIG. 1. Thedistribution of VL gene has been found to have a bias toward someparticular genes. This result demonstrates that a high-quality librarycontaining functional active immunoglobulins can be prepared byselecting a set containing, in a high percentage, many light chainsallowing the re-holding of the immunoglobulin conformation.

In this step, it is preferred that as many amino acid sequences aspossible are analyzed to mimic the in vivo antibody diversity.

In the human genome, genes constituting the light chain include 36 typesof Vλ, 7 types of Jλ, 37 types of Vk, and 4 types of Jk. Since acombination of V gene and J gene produces a light chain gene, a simpleestimate is as follows: summation of (36×7=252) and (37×4=148), namely400 types of vitiations. In addition, mutational events occur duringjoining genes, and thus further increase the number of amino acidvariations at the junction. Specifically, the event specific to antibodygene rearrangement as described above produces variations of about ±1amino acid in average (about ±5 amino acid at most). After somedispensable genes are further eliminated from the combinations, therepertoire is completed in an individual. In addition, there are minorvariations in the genes in each individual (which is referred to as“polymorphism”). It is practically impossible to study the whole typesof antibody genes of the entire human beings, but the total number hasbeen estimated to be 1,000. These findings shows that, when a humanindividual can produce a set of antibodies corresponding to all types ofantigens, the repertoire for the light chain variable region gene can bereproduced successfully by analyzing theoretically preferably about 400to 1,000 types of amino acid sequences by the method of the presentinvention.

The repertoire size for the human light chain (200 types) was estimatedby the present inventors based on the result obtained by analyzingapproximately 1,000 types of amino acid sequences. Thus, theoretically,it can be assumed that the light chain variable region genes areselected so as to allow the re-holding of functionally activeconformation in every antibody set. The present invention hasexperimentally proven that the repertoire size of light chain variableregion genes determined to be 200 types according to the presentinvention is large enough to mimic in vitro the in vivo antibodydiversity. However, there is a possibility that the repertoire size isdetermined to be larger by analyzing amino acid sequences deduced frommuch more nucleotide sequences.

Then, the repertoire size of the library of light chain variable regiongenes selected according to the present invention can be increased bycombining the library with another light chain gene library. Namely, alibrary of light chain variable region genes (referred to as VL library)is prepared by using a non-biased collection of the entire light chaingenes without selection by the same method used for the heavy chain. Alibrary whose shortage has been supplied by combining the VL libraryprepared as describe above with the library (referred to as KL200library) comprising only 200 types of light chain variable region genes.The respective libraries have the characteristics described below.

KL200 library has been confirmed to comprise the light chains allowingthe re-holding of the functionally active conformation when combinedwith the heavy chain. However, there is a possibility that light chainsrequired for clones having particular specificities have been excludedfrom the library because of the limited number.

VL library covers all of the clones required, because the number ofindependent clones in it is 10⁹. However, the level of expression andthe rate of conformation formation with the heavy chain are lower thanthose of KL200 library.

Basically, genes can be selected arbitrarily from each group classifiedbased on the analysis result of amino acid sequences according to thepresent invention. Thus, it is not quite significant to identify thestructures of genes encoding light chains allowing the re-holding offunctionally active conformation in combination with the heavy chain.The most important is to practice the selection step for the lightchains that allows the re-holding of functionally active conformation incombination with the heavy chain according to the selection method. Bythe procedure, a human light chain variable region gene library can beprepared from a library comprising 101 types of κ chain genes andanother library comprising 99 types of λ chain genes.

As described below, when combined with genes encoding the heavy chainvariable region, the library of the light chain variable region genesselected according to the present invention provides an immunoglobulingene library. Thus, the library of light chain variable region genesselected according to the present invention can be used to prepare animmunoglobulin gene library. Specifically, the present invention relatesto a gene library consisting of at least genes encoding the light chainvariable region of immunoglobulin, in which genes encoding the lightchain variable domains incapable of re-holding functionally activeconformation when combined with the immunoglobulin heavy chain have beensubstantially eliminated.

In the present invention, the genes encoding the light chain variableregion incapable of re-holding functionally active conformation whencombined with the immunoglobulin heavy chain can be eliminated by theselection method for the light chain variable region as described above.A gene encoding the light chain variable region incapable ofre-holding-functionally active conformation when combined with theimmunoglobulin heavy chain is herein referred to as “defective gene”. Inthe present invention, a library where defective genes have beensubstantially excluded does not refer to a library from which defectivegenes have been eliminated completely. For example, a library wheredefective genes have been contaminated can be assumed to be a librarywhere defective genes have been substantially excluded, when thepopulation of contaminated genes falls within a range where the antibodyscreening based on immunological reaction is not prevented.

The “range where the antibody screening based on immunological reactionis not prevented” means that the percent population of defective genesin an antibody library ranges, for example, of 0 to 50%, preferably 0 to25%. As a matter of course, the smaller the population of defectivegenes, the higher the screening efficiency and the lower the risk forthe loss of useful clones during the screening step. However, theinventors prepared trial libraries and tested the screening efficiency;a VL library in which 50% of the genes are assumed to be defective geneswas combined at various ratios with a KL200 library, from whichdefective genes had been eliminated completely; then, it was confirmedthat effective screening was secured up to 1:1 ratio. This fact showsthat defective genes can be assumed to be substantially excluded whenthe percent rate of defective genes is more preferably 25% or less.

A library consisting of only the light chains of the present inventionis useful as material to be combined with a library of genes encodingthe heavy chain variable region. Such a library includes, for example, aphage library in which a light chain library from which defective geneshave been substantially excluded have been inserted into the geneencoding cp3 protein of phage, and the vector of which has a cloningsite to insert the heavy chain variable region gene. Typically, methodsfor preparing phage libraries comprise inserting a foreign gene in thephagemid so that the phage could retain the infectivity to the hostmicroorganism and packaging the phagemid into a phage particle by usinghelper phage. The phage library of the present invention can also beconstructed by using a phagemid. Specifically, a cloning site, at whichis to be used for inserting a gene encoding the above-mentioned lightchain variable domain connected with a signal sequence and a geneencoding the heavy chain variable domain, is placed downstream of apromoter which is operative in the host. The cloning site for the heavychain variable region gene advantageously contains a site recognizableby a restriction enzyme which digests genes of interest in a minimalfrequency. The phage library of the present invention can be preparednot only using the phagemid but also using phage genome. The heavy chainvariable region gene is synthesized by PCR using primers additionallycontaining cloning site, and then inserted into the phage library; thusa phage library for the expression of immunoglobulin variable region canbe completed.

Alternatively, an E. coli strain that can express and secrete a lightchain variable domain can be prepared by inserting a light chain libraryfrom which defective genes have been substantially eliminated into an E.coli expression vector and transforming E. coli cells with the lightchain variable region library. Phage particles on which Fab has beenre-folded on the surface can be prepared by infecting to E. coli thephages in which the heavy chain variable region genes obtained by PCRhave been inserted. An antibody library containing desired antibodiescan be prepared by selecting heavy chain variable region genes from avariety of individuals having different immunological histories.

A kit for preparing a phage library can be provided based on such amethod. Such kits comprise a light chain gene library from whichdefective genes have been substantially excluded and primers foramplifying the heavy chain variable region genes. Users can prepare PCRproducts from a gene source having an immunological history, which canprovide genes encoding desired antibodies, using primers ensuring theamplification of heavy chain variable region genes. For example, alibrary in which a population of antibodies recognizing tumor-associatedantigens has been enriched can be obtained from a host affected withcancer.

A library of the present invention is prepared by using the light chainvariable region genes selected as described above. Specifically, thepresent invention relates to a method for preparing a gene librarycomprising combinations of light chain variable region genes and heavychain variable region genes of immunoglobulin, which comprises thefollowing steps:

(a) selecting a light chain variable region genes encoding light chainmolecules capable of re-holding functionally active conformation, inconjunction with expressed product of heavy chain variable region genes;

(b) constructing a gene library which is a collection of light chainvariable region genes obtained in step (a); and

(c) combining the library obtained in step (b) with a library of genesencoding the heavy chain variable region.

The selection for the light chain in step (a) is as described above. Thelibrary of step (b) can be prepared by collecting previously obtainedgenes encoding the light chain. When the light chain variable regiongenes are introduced in filamentous phage particles, a library can beobtained by recovering the phage particles amplified. Then, in step (c),the above-mentioned light chain gene library is combined with a heavychain gene library. The heavy chain variable region genes can beobtained from antibody-producing cells such as peripheral bloodlymphocytes and splenocytes by a method known to those skilled in theart. For example, there are seven VH families of VH1 to VH7 for humanimmunoglobulin.

Primers ensuring the amplification of respective genes belonging to eachfamily are known to those skilled in the art (Campbell, M. J., Zelenetz,A. D., Levy, S. & Levy, R. (1992). Use of family-specific primers forPCR amplification of the human heavy chain variable gene repertoire.Mol. Immunol., 29, 193-203; in addition, such primers are publicized onthe homepage of MRC Co. “V-base”). Thus, the heavy chain variable regiongenes can be amplified for each family by RT-PCR using such primers.

The heavy chain variable region genes obtained as amplification productscan be converted to a gene library by inserting each gene into anappropriate vector. In this step, the library of the heavy chainvariable region genes is prepared separately for each VH family; therespective libraries are combined together in accordance with the invivo ratio of the respective families; thus, the gene library of thepresent invention can mimic the in vivo antibody repertoire.Specifically, for example, in human, the respective populations areroughly estimated to be at the following ratio. Mimicking the in vivoantibody repertoire can reduce the chance of losing desired clonesduring screening.

VH1: 25%

VH2: 6.6%

VH3: 40%

VH4: 19%

VH5: 5%

VH6: 3.8%

VH7: 1.2%

The preparation of heavy chain variable region genes is described belowin more detail. Primers are designed for the respective seven VHfamilies, and then RT-PCR is carried out using the primers incombination with a primer common to six types of JH genes. Primers thatenable to amplify a wide range of genes of each human VH family areknown to those skilled in the art (Marks J. D. et al., J. Mol. Biol.(1991) 222, 581-597; Campbell, M. J., Zelenetz, A. D., Levy, S. & Levy,R. (1992); Use of family-specific primers for PCR amplification of thehuman heavy chain variable gene repertoire. Mol. Immunol., 29, 193-203;in addition, such primers are publicized on the homepage of MRCCorporation, “V-base”). It should be confirmed that exact genes of eachfamily are amplified corresponding to the primers used. Specifically,dozens of clones corresponding to bands of amplified products having theVHDJH structure are isolated from each family, and then their nucleotidesequences are determined to analyze which heavy chain variable regiongene was amplified. When some genes are hardly amplified, extra primersare newly designed and added. For example, it has been reported thatsome new primers allow the amplification of genes that cannot beamplified by conventional primers.

When having the VHDJH structure in frame, each of such clones areinserted into an appropriate vector to analyze the expression level inE. coli, holding with a protein encoded by the light chain variableregion gene, and folding. When any step is poorly achieved with someclones, then one should deduce the reason and estimate the percentpopulation of such clones in the library.

The population of each immunoglobulin molecule in an individual dependson the immunological history. Thus, it is important to prepare heavychain variable region genes from a wide variety of B cells so as toreflect as many immunological histories of individuals as possible. Inpractice, the number of types of gene source available for the heavychain variable region genes should be increased, e.g., umbilical blood,tonsil, peripheral blood, bone marrow, etc.

Further, it is also important to prepare B cells from a naive B cellpopulation which has no experience of contacting with immunogens(including autoantigens). Because clones recognizing self antigens areeliminated during the maturation period of immune system. Naive B cellsare important to construct a gene library further containing arepertoire of antibodies against autoantigens. Such libraries arecarefully combined so that the number of clones is proportional to thenumber lymphocytes. The VHDJH library eventually prepared should containindependent clones on the order of 10⁹ (10⁹ to 10¹⁰).

The treatment has the significance as described below. The amino acidsequence constituting antigen-binding surface of an antibody isdetermined by the genes on the genome for light chain CDR1, CDR2, andCDR3, and heavy chain CDR1 and CDR2 (including evolutionary selection);the entire variations cover about 10,000 types. An enormous number ofheavy chain CDR3 variations further increase the level of diversity. Itis necessary that the antibody library contains the 10,000 variations asequally as possible and variations of heavy chain CDR3 (which isproduced in a random process in each B cell of each individual)maximally. The above-mentioned method can meet this demand.

By the above-mentioned analyses, the present inventors found that the VHgene was not exactly expressed when a CDR contained cysteine residues.They also confirmed that 70% or more clones in the prepared heavy chainvariable region library were successfully expressed, holded with theheavy chain variable region domain, and folded in E. coli.

Alternatively, the library can be characterized roughly by selecting asource for heavy chain variable region genes based on the immunologicalhistory. For example, the probability that immunoglobulin exhibitinghigh affinity for the pathogen is obtained is higher, when the personhas a previous history of an infectious disease. When antibody-producingcells from a cancer patient are used as a source for the heavy chainvariable region genes, immunoglobulins recognizing a tumor-associatedantigen may be obtained.

Further, artificial mutations introduced into the VH genes can increasethe number of variations in a library. A method, known to those skilledin the art, for introducing artificial mutations is error-prone PCR(Winter, G. et. al., Annu. Rev. Immunol., 12, 433-455, 1994). Sincedefective genes for light chain variable domain have been substantiallyexcluded from the library of the present invention, the diversity of theheavy chain variable region gene directly contributes to the diversityof the library. Thus, such a library can attain a diversity of extremelyhigh level simply by carrying out error-prone PCR which is known tothose skilled in the art. The error-prone PCR can be practiced asfollows.

The error-prone PCR is a method for introducing random point mutations.Specifically, one can utilize the following biochemical properties ofDNA polymerase to be used in PCR.

(1) While typically Taq DNA polymerase is used in the presence of Mg²⁺ion, the fidelity of the enzyme for nucleotide incorporation is impairedin the presence of Mn²⁺ ion.

(2) While typically the nucleotide monomers, dATP, dCTP, dTTP, and dGTP,are used at an equal concentration in PCR, their concentrations can bealtered so as to ensure the introduction of mutations at a higher rate.

(3) dITP is also used in addition to the four types of nucleotidemonomers. By Taq DNA polymerase, dITP is incorporated as inosinenucleotide into a DNA chain. No base pair is formed between an inosineresidue and any of the four nucleotides; as PCR proceeds, at theposition complementary to an inosine, any one of the five types ofnucleotides can be incorporated at random.

Mutations can be introduced at random based on the synergistic effect ofthe above-mentioned three factors. Specifically, the reaction can becarried out under a condition, for example, of 7.5 mM MgCl₂, 0.5 mMMnCl₂, 0.2 mM dATP/dGTP, 1.0 mM dCTP/dTTP, 0.1 to 1.0 mM dITP. Inpractice, conditions, such as a concentration, are further adjusted tomeet the experimental purpose. Reaction conditions such as temperaturemay be the same as used in typical PCR experiments.

Vectors known to those skilled in the art can be used for preparing agene library of the present invention. Vectors that can be used in thepresent invention include, for example, a phage library containing thelight chain variable region gene library prepared as described above.Specifically, a heavy chain variable region gene is inserted upstream ofthe light chain variable region gene in a phagemid (Iba, Y. et al., Gene194 (1997) 35-46). A phage library in which the phages express the heavychain variable region and light chain variable region at the same timeon their surface can be used advantageously to practice the panningmethod (see below) using the antigen binding as an index.

Specifically, an rgdp library can be provided by preparing the genelibrary of the present invention as a phage library. The gene library ofthe present invention can be prepared as an rgdp library using a systemwhere a foreign gene is expressed as a fusion protein with a ribosomeprotein or a protein constituting E. coli flagella as well as a phageprotein.

A representative rgdp library is the phage library. A phage librarybased on the antibody library of the present invention can be preparedas described below. Phagemid or helper phage is generally used toexpress a foreign protein on phage surface. For example, phagemidvectors such as pTZ19R (Pharmacia) are commercially available. When aphagemid is used, the gene encoding a foreign protein to be expressed isligated with a gene encoding phage protein cp3, cp8, or the like.

A phagemid can be amplified by infecting a host such as E. coli with it.However, phage particles cannot be recovered by only this treatment. Ina word, the state after the treatment as described above is the same asthat of a typical gene library. Superinfection of helper phage to themicroorganism already infected with phagemid allows the display of theforeign protein, whose gene has been inserted in the phagemid, on thesurface of phage particles. For example, phage particles for thephagemid vector pTZ19R can be recovered upon superinfection of helperphage M13K07. When the foreign protein is fused with cp3 protein derivedfrom the phagemid used, the foreign protein is displayed on the surfaceof the resultant phage particles.

When a restriction enzyme site suitable for the cloning of an antibodygene has been introduced into a commercially available phagemid, thelight chain variable region gene library of the present invention incombination with a heavy chain variable region gene library can beinserted into it. A method, which comprises introducing an appropriaterestriction enzyme site into the phagemid vector pTZ19R and inserting anantibody gene library amplified by PCR into it, is known to thoseskilled in the art (Gene 194, 35-46, 1997). In Example described below,SfiI site and AscI site were introduced downstream of the signalsequence PelB into a phagemid vector. On the other hand, a primer havingthe same restriction enzyme site is used to amplify the light chainvariable region gene. The amplification products obtained by PCR areinserted at the restriction enzyme site which has been pre-cleaved withthe enzyme; the antibody variable region gene is thus placed downstreamof PelB. The constructed phagemid vector encodes a antibody variableregion protein fused with cp3 located further downstream (pFCAH9-E8d inFIG. 2).

The heavy chain variable region gene can also be inserted into anexpression vector for a bacterial host. In this case, the heavy chainvariable region gene can be expressed by transforming bacterial cellswith the vector. A gene library of the present invention is finallycompleted by further infecting a phage library containing the lightchain variable region gene to the transformed cells obtained. Thevectors for E. coli transformation include pFK, etc. In cases wherebacterial cells are transformed with the phages, when the heavy chainvariable region gene has been inserted downstream of an appropriatesecretory signal, the heavy chain variable region protein can besecreted into periplasm. pFK is a vector containing the secretory signalpelB.

Further, the library of the present invention can also be prepared byinserting the light chain variable region gene library prepared asdescribed above and a library of heavy chain variable region genes intoan expression vector for animal cells. Such vectors include pcDNAI, etc.

The nucleotide sequence encoding a label peptide can previously be fusedto the 3′ end of heavy chain variable region genes constituting the genelibrary of the present invention. Such label peptides include, forexample, histidine tag (His×6), myc-tag, HA-tag, etc. The histidine taghas metal ion-binding activity. When a heavy chain constituting thelibrary of the present invention has been fused with the histidine tag,for example, phage particles expressing the heavy chain can be trappedin a nickel column. Clones expressing the heavy chain can be enrichedand purified by washing off the particles of the phage and helper phageunbound to the nickel column.

The gene library of the present invention may be any type of antibodylibrary. Specifically, such libraries include phage library, E. colilibrary, ribosome library, etc. The antibody library according to thepresent invention can be prepared by expressing the immunoglobulin genesin the clones constituting the antibody library according to the presentinvention and recovering the expression products. The present inventorshave found that when E. coli infected with a phagemid containing, as aninsert, the light chain variable region gene and heavy chain variableregion gene fused with the cp3 gene, is cultured without adding helperphage for a long period, the fusion protein of Fab and cp3 is secretedinto the culture supernatant from E. coli. The characteristics ofantibody gene in a phagemid can readily be tested by utilizing thisphenomenon. Further, each clone is analyzed to confirm whether there-holding has been achieved between the light chain variable domain andheavy chain variable domain, and such analysis allows excluding clonesfailing the re-holding. The antibody library of the present inventioncan mimic the in vivo antibody population by this treatment.

The antibody library of the present invention can be used to screen fordesired variable region genes through screening the antibody. With theantibody library of the present invention, without distributing thevariable region gene library itself, one can provide only the antibodylibrary to a third party who practices the screening for desiredantibody. The present invention relates to a method for detectingantibodies having desired reactivity in the library of the presentinvention. The antibody reactivity can be evaluated by theantigen-binding activity. Specifically, desired antibodies can bedetected using as an index the activity of binding to an antigen ofinterest by the following steps:

(a) contacting the antigen with the antibody library according to thepresent invention under a suitable condition for antigen-antibodyreaction; and

(b) detecting the binding between the antigen and the immunoglobulinvariable domain.

Further, the present invention relates to a method for obtainingimmunoglobulin variable domain binding to a specific antigen by usingthe library of the present invention. Firstly, the method of the presentinvention for detecting antibody variable domain comprises the stepdescribed below.

Further, the method of the present invention for obtainingimmunoglobulin variable domain which binds to a particular antigenfurther comprises the following step (c) in addition to theabove-mentioned steps (a) and (b):

(c) selecting a clone expressing the immunoglobulin variable domainwhich binds to the above-mentioned antigen.

The library to be used in step (a) can be a library that ensures theexpression of both light chain variable domain and heavy chain variabledomain, or an antibody library comprising the expression products fromthe former library. In a particularly useful library, fragments of theheavy chain and light chain encoded by the genes constituting thelibrary are expressed on the surface of filamentous phage, and they arere-holding the functionally active conformation on the surface of phageparticles. This type phage library serves as an rgdp library, and withthis library, clones having desired reactivity can readily be enrichedby the panning method. The panning method according to the presentinvention can be practiced as follows.

First, an antigen of interest is contacted with an rgdp library, andthen clones bound to the antigen are recovered. After being amplified,the recovered clones are again contacted with the antigen of interest.Then, clones bound to the antigen are recovered. This cycle is furtherrepeated. The amplification of clones is achieved by infecting E. colicells with phage and recovering the phage particles. The variable domainhaving desired reactivity is enriched by repeating this step. Ingeneral, such screening based on the antigen-binding activity isrepeated until the recovery rate of clone is markedly increased. The“recovery rate” used herein refers to a ratio of: (the number ofrecovered clones having the antigen-binding activity) to (the number ofclones charged with an antigen-coated plate). When the recovery rate ismarkedly increased as compared with that of the previous round ofscreening, phage particles displaying the antibodies having desiredreactivity are assumed to be enriched.

In addition, screening can be carried out by using an antibody librarythat comprises a collection of separately recovered expression productsof the respective clones constituting a library. It is also possible todirectly select immunoglobulin having desired reactivity by contactingthe expression product of each clone with an antigen of interest. Adesired clone for the variable region can be obtained by selecting aclone encoding immunoglobulin that has been detected to bind to anantigen.

In the present invention, any compound having epitopes can be used as anantigen to detect and obtain an antibody variable domain. It has beenknown that a wide variety of proteins, sugars, nucleic acids, organiccompounds, and inorganic compounds have epitopes. These compounds may bederived from biological samples or synthesized artificially.Specifically, such compounds that can be used as antigens include thoseof animals and plants; microorganisms such as bacteria and fungi; cellsand particles such as viral particles; proteins constituting them; sugarchains; lipids, etc. Further, various nonproteinous agents, hormones,vitamins, cytokines, chemokines, chemical substances causingenvironmental pollution, and others can be used as antigens in thepresent invention, when having epitopes.

Further, the whole antigen molecule that is a target of a desiredantibody or a part of the antigen molecule may be used as an antigen.Unless otherwise specified, the antigen of the present invention alsoincludes compounds comprising a partial structure of the antigen. Forexample, a high-specificity antibody can be obtained by using a partialantigen molecule containing an epitope comprising a specific structureof the antigen molecule.

Further, a complex of multiple molecules can be used as an antigen.Unless otherwise specified, the antigen of the present invention alsoincludes a complex of multiple molecules. Using such a complex as anantigen, it is possible to obtain an antibody that can discriminate thecomplex from each monomeric molecule.

For example, an antibody molecule that can be used as a neutralizingantibody against a pathogen can be detected in the library of thepresent invention, which can be followed by isolation of the geneencoding the same from the library. In the present invention, thepathogen includes every pathogenic organism, and every substance derivedform such an organism. More specifically, such organisms include virus,bacterium, fungus, mycoplasma, multi-cellular parasitic organism, etc.The pathogen of the present invention also includes toxins derived frommicroorganisms, animals, and plants. Thus, the pathogen of the presentinvention also includes toxins produced by cholera vibrio andenterohaemorrhagic Escherichia coli, etc. The pathogen of the presentinvention also includes biotoxin such as snake venom, bee toxin, etc. Onthe other hand, the neutralizing antibody of the present inventionrefers to an antibody having the activities of suppressing pathogenicityand infectivity of the pathogen.

The method of the present invention for detecting such a neutralizingantibody comprises detecting a clone expressing the immunoglobulinvariable domain binding to the antigen by contacting the antigen derivedfrom the pathogen with the library of the present invention. Further,the present invention provides a method for screening for a neutralizingantibody by selecting a clone expressing the variable domain binding tothe antigen.

In the method of the present invention for screening for a neutralizingantibody, antibodies comprising the variable region having undesiredreactivity can be removed from the antibody library by absorption. Forexample, it has been believed that antibodies against the HA antigen ofinfluenza virus are effective to suppress the infectivity. On the otherhand, among antibodies against the same influenza virus particles,antibodies against the nuclear protein (NP) have no virus-neutralizingactivity. However, when the influenza virus particle is used as anantigen, anti-NP antibodies are often detected. As a result, screeningefficiency for neutralizing antibodies may be impaired. In such cases,when anti-NP antibodies have been pre-absorbing, efficient detection ofanti-HA antibodies can be achieved. Such anti-NP antibodies can beabsorbed by contacting the library of the present invention with the NPantigen of influenza virus particles under a condition allowing theantigen-antibody reaction, and removing antibody variable region boundto NP antigen. Such antibodies against HA antigen can be preparedefficiently, for example, by using, as an index, the binding activity topurified HA antigen sample containing no NP antigen. However, the methodcomprising the step of pre-absorbing anti-NP antibodies can be usedadvantageously to confirm the reactivity of an antibody against virusparticles having the original surface structure.

The genes obtained as described above, which encode immunoglobulinvariable domains, and the immunoglobulin variable domains that are theexpression products are within the scope of the present invention. Theisolated immunoglobulin variable domains can be used for diagnosing andtreating diseases. In particular, when the antibody is a humanimmunoglobulin, it can be administered into the human body.Specifically, such an antibody can be used for diagnosing and treatingvarious infectious diseases, tumor, arteriosclerosis, etc. Variousdiagnostic and therapeutic methods using immunoglobulin are known tothose skilled in the art.

When a perfect human immunoglobulin molecule is prepared from theimmunoglobulin variable domain obtained according to the presentinvention, it can be used not only as a simple affinity ligand but alsoas an antibody molecule. Specifically, the heavy chain variable regiongene and light chain variable region gene obtained according to thepresent invention are ligated with CH gene and CL gene encoding theconstant regions, respectively. When genes encoding the constant regionsderived from IgG are used, the resultant immunoglobulin molecule canhave an excellent opsonin activity.

All publications describing prior art cited herein are incorporatedherein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram summarizing the classification of VL genes ofphage antibodies obtained according to the present invention.

Abscissa: 63 types of light chain variable region genes from germline(chromosome).

Ordinate: Types of antigens used in the screening for phage antibodies

From the deepest:

1. Tetanus toxin (TET)

2. Influenza virus antigen (IFL)

3. varicella-zoster virus antigen (VZGH)

4. Diphtheria toxin (DTD)

5. Summation for the items 6 and 7 (total)

6. Summation for sensitizing antigens (1+2+3+4) (immunized)

7. Summation for non-sensitizing antigens (antigens which ordinarypersons never encounter in their lifetime, such as C. elegans) (noimmunized)

Height: the number of phage antibodies.

FIG. 2 shows a schematic illustration of structures of various vectorsused for constructing the variable region library of the presentinvention.

(1) pAALFab: vector for D1.3 mutation.

(2) pFCAH3-E8T: expression vector for E8. This vector was constructed bymodifying the restriction enzyme sites based on pAALFab. PstI, XbaI, andKpnI sites have been newly added; positions of the EcoRI and XhoI siteshave been changed.

(3) pFvCA-E8VHd: cloning vector for the heavy chain variable regiongene. This vector was constructed by modifying the restriction enzymesites based on pFCAH3-E8T. The XbaI-EcoRI portion has been deleted;KpnI, SfiI, NcoI, and SpeI sites have been newly added. A heavy chainvariable region gene can be cloned between SfiI and XhoI sites.(4) pFCAH9-E8d: cloning vector for the heavy chain variable region gene.This vector was constructed by modifying the DNA sequence based onpFCAH3-E8T and pFvCA-E8VHd. Human γCH1 has been substituted for mouseγCH1. SfiI, NcoI, and AscI sites have newly been added. Alight chainvariable region can be cloned between the SfiI and AscI sites.

FIG. 3 shows the nucleotide sequence of insert in pFCAH9-E8d (SEQ ID NO:99).

FIG. 4 shows positions of restriction enzyme sites in the nucleotidesequence (SEQ ID NO: 99 of the insert in pFCAH9-E8d and the amino acidsequence encoded by the nucleotide sequence (1) (SEQ ID NO: 100).

FIG. 5 shows positions of restriction enzyme sites in the nucleotidesequence (SEQ ID NO: 99) of the insert in pFCAH9-E8d and the amino acidsequence encoded by the nucleotide sequence (2) (SEQ ID NO: 101).

FIG. 6 shows positions of restriction enzyme sites in the nucleotidesequence (SEQ ID NO: 99) of the insert in pFCAH9-E8d and the amino acidsequence encoded by the nucleotide sequence (3) (SEQ ID NO: 102).

FIG. 7 shows the nucleotide sequence of the insert in pscFvCA-E8VHd (SEQID NO: 103).

FIG. 8 shows positions of restriction enzyme sites in the nucleotidesequence (SEQ ID NO: 103) of the insert in pscFvCA-E8VHd and the aminoacid sequence encoded by the nucleotide sequence (1) (SEQ ID NO: 104).

FIG. 9 shows positions of restriction enzyme sites in the nucleotidesequence (SEQ ID NO: 103) of the insert in pscFvCA-E8VHd and the aminoacid sequence encoded by the nucleotide sequence (2) (SEQ ID NO: 105).

FIG. 10 shows a result obtained by assaying the affinity of theantibodies selected from the antibody library according to the presentinvention. The ordinate indicates absorbance at 492 nm; the abscissaindicates clone number. PC in the abscissa refers to a positive control.

FIG. 11 shows the reactivity of each clone listed in Table 15 to gH orgE, which was measured by ELISA.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is illustrated in detail below with reference toExamples.

Example 1 Preparation of Phagemid Vectors for Library Construction

1-1 Preparation of Vectors to Construct Combinatorial Libraries of theHeavy Chain and Light Chain

As shown schematically in FIG. 2, the vector pFCAH9-E8d was prepared byinserting M13 phage-derived pelB (signal sequence), His6 tag sequence(SEQ ID NO: 106), M13 phage-derived sequence encoding cp3 protein (Acp3(198aa to 406aa): the capsid protein 3 lacking the N terminus), and DNAencoding the amino acid sequence of protein A at appropriate restrictionenzyme sites into pTZ19R phagemid vector (Pharmacia) (see Iba, Y. et al.GENE 194 (1997) 35-46). The genes encoding light chains λ5 and λ6contained BstPI sites; to avoid such cleavage, pFCAH9-E8d has beendesigned to contain an XhoI site in addition to a BstPI site. Thenucleotide sequence of the insert in pFCAH9-E8d is shown in FIG. 3; therestriction enzyme sites and the amino acid sequence encoded by thenucleotide sequence are shown in FIGS. 4 to 6.

A vector directing the expression of an antibody protein is completed byinserting the heavy chain and light chain genes at desired positionsinto the vector described above. With the vector constructed, theantibody is expressed as a Fab-type antibody; each of heavy chain andlight chain contains the variable region at the N-terminus which isfollowed by the constant region CH1 or CL. The heavy chain is linkedwith the light chain via a disulfide bond between the constant regions.The gene CL encoding the light chain constant region is fused with theabove-mentioned cp3 gene, and as a result the protein expressed is aFab-cp3.

Specifically, the procedures used are as follows:

Primers used:

527 Reverse (SEQ ID NO: 2): 5′-CAGGAAACAGCTATGAC-3′599 E8VHf-PstR (SEQ ID NO: 3): 3′-CGGCTCCAAGTCGACGTCGTCA-5′544 E8VHf-PstF (SEQ ID NO: 4):5′-CAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAA-3′545 E8VHf-XbaR (SEQ ID NO: 5):3′-AGACCGAAGTTGTAATTTCTGTGGATATACGTGACCCACTTCGTCTCCGGACTTTTCCCAGATCTCACCTAACCTTCCTAA-5′ 546 E8VHf-XbaF (SEQ ID NO: 6):5′-AAGGGTCTAGAGTGGATTGGAAGGATTGATCCTGCGAGTGGTAATACTAAATATGACCCGAAGGACAAGGCCACTATAACAGCA-3′547 E8VHf-EcoR (SEQ ID NO: 7):3′-TTCCTGTTCCGGTGATATTGTCGTCTGTGTAGGAGGTTGTGTCGGATGGATGTCGACTTAAGGGAC-5′ 548 E8VHf-EcoF (SEQ ID NO: 8):5′-CAGCTGAATTCCCTGACATCTGAGGACACTGCCGTCTATTACT GTGCTGGT-3′549 E8VHf-BstR (SEQ ID NO: 9):3′-CAGATAATGACACGACCAATACTAATGCCGTTGAAACTGATGACCCCGGTTCCGTGGTGCCAGTGGCACAAGG-5′ 590 His6-SmaR (SEQ ID NO: 10):3′-GGTTCTCTAACAGTAGTGGTAGTAGTGGTAATTATTCTCGATA GGGCCCTCGAA-5′542 E8VLf-SacF (SEQ ID NO: 11):5′-GACATCGAGCTCACCCAGTCTCCAGCCTCCCTTTCTGCGTCTGTGGGAGAAACTGTCACCATCACATGT-3′ 539 E8VLf-KpnR (SEQ ID NO: 12):3′-TGACAGTGGTAGTGTACAGCTCGTTCACCCTTATAAGTGTTAA TAAATCGTACCATGGTCGTC-5′542 E8VLf-KpnF (SEQ ID NO: 13):5′-GCATGGTACCAGCAGAAACCAGGGAAATCTCCTCAGCTCCTGG TCTAT-3′543 E8VLf-BamR (SEQ ID NO: 14):3′-GGAGTCGAGGACCAGATATTACGTTTTTGGAATCGTCTACCACACGGTAGTTCCAAGTCACCGTCACCTAGGCCTTGTGTT-5′562 E8VLf-XhoR (SEQ ID NO: 15):3′-TCATGAGGCACCTGCAAGCCACCTCCGTGGTTCGAGCTCTAGT TT-5′563 E8VLf-XhoF (SEQ ID NO: 16):5′-AGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTCGAGATCA AA-3′613 NheR (SEQ ID NO: 17): 3′-ATCGACAGCT-5′600 E8VLKpnXhoR (SEQ ID NO: 18): 3′-AAGCCACCTCCATGGTTCGAGCTCTAGTTT-5′LCP3ASC (SEQ ID NO: 19): 3′-TCGAAGTTGTCCTTACTCACAAGCCGCGCGGTCAGCTGAGGTAA-5′ hCH1Bst (SEQ ID NO: 20):5′-ACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCG GTCTTCCCCCTGG-3′hCH1midAS (SEQ ID NO: 21): 3′-GGGAGTCGTCGCAGCACTGGCACGGGAGGTCGTCGAA-5′hCH1midS (SEQ ID NO: 22): 5′-GGACTCTACTCCCTCAGCAGCGTCGTGACCGTGCCC-3′hCH1H6 (SEQ ID NO: 23): 3′-GGGTCGTTGTGGTTCCACCTGTTCTTTCAACTCGGGTTTAGAACAGTAGTGGTAGTAGTGGTA-5′ hCH1H6Sma (SEQ ID NO: 24):3′-GGGTTTAGAACAGTAGTGGTAGTAGTGGTAATTATTCTCGAT AGGGCCCTCGAACG-5′702 BstXhoF (SEQ ID NO: 25): 5′-GGCACCACGGTCACCGTCTCGAGCGCCTCCACC-3′Preparation of the Heavy Chain Region in pFCAH3-E8T(1) DNA fragments were prepared by PCR using pAALFab as a template andprimers 527-599 or primers 547-590.(2) DNA fragments were prepared by PCR using primers 544-545, primers546-547, or primers 548-549.(3) The PCR products obtained in (1) and (2) were combined together.Then, PCR was carried out using primers 527 and 590, and the productswere cloned into pAALFab at the HindIII-Sinal site.pFCAH3-E8T Light Chain Region(4) DNA fragments were prepared by PCR using primers 542-562, or primers561-613.(5) DNA fragments were prepared by PCR primers 538-539, or primers542-543.(6) The PCR products obtained in (4) and (5) were combined together.Then, PCR was carried out using primers 538 and 562, and the productswere cloned into pAALFab at the SacI-NheI site.pfCAH9-E8d(6) Preparation of VH stuffer regionpFCAH3-E8T was double-digested with XbaI and EcoRI, and both ends wereblunted with klenow fragment. Then, the vector was self-ligated toprepare VH stuffer.(7) Preparation of VH stuffer regionPCR was carried out using pFCAH3-E8T as a template and primers 527-600.The PCR products were cloned at HindIII-XhoI site into the constructobtained in (6).(8) The constructed DNA was digested with KpnI, and then self-ligated toprepare VL stuffer.(9) Introduction of SfiI, NcoI, and SpeI sitesPCR was carried out using pFCAH3-E8T as a template and primers 527-663.The PCR products were cloned at HindIII-SacI site into the constructobtained in (1).(10) Introduction of AscI sitePCR was carried out using pFCAH3-E8T as a template and primers527-LCP3ASC. The PCR products were cloned into the construct obtained in(2) that had been completely digested with SacI and partially digestedwith SalI.(11) Replacement of the gamma CH1 region with the human geneThe human gamma CH1 region has BstPI sites; the gene was cloned with astrategy to abolish the BstPI sites. PCR was carried out using a cDNAderived from the tonsil as a template and primers hCH1Bst-hCH1midS orprimers hCH1midAS-hCH1H6. The PCR products were combined together; PCRwas carried out using the mixture as a template and primershCH1Bst-hCH16Sma. The DNA fragment was cloned at BstPI-Sma site into theconstruct obtained in (3).(12) Introduction of Xho sitePCR was carried out using the construct obtained in (11) as a templateand primers 702-663, and the products were cloned at BstPI-SacI siteinto the construct obtained in (11).1-2 Preparation of Vector to Transiently Clone the Heavy Chain VariableRegion

First, the pAALFab vector (FIG. 2) was constructed according to a methodknown to those skilled in the art (see Iba, Y. et al., GENE 194 (1997)35-46). The XbaI-EcoRI fragment was deleted from the pAALFab vector, andrestriction enzyme digestion sites KpnI, SfiI, NcoI, and SpeI were newlyadded to the vector. The vector pscFvCA-E8VHd (FIG. 2) allowing thecloning of VH (heavy chain variable region) was finally constructed viapFCAH3-E8T. The vector constructed was used as a vector to transientlyclone the heavy chain variable region. The nucleotide sequences of theinserts in pscFvCA-E8VHd are shown in FIG. 7, and restriction enzymesites and the amino acid sequences encoded by the nucleotide sequencesare shown in FIGS. 8 and 9.

Specifically, the procedures are as follows:

Primers used:

610 scBstSpeSacF (SEQ ID NO: 26):

610 scBstSpeSacF (SEQ ID NO: 26):5′-CACCACGGTCACCGTCTCCTCAGGCGGTGGCGGATCAGGTGGCGGTGGAAGTGGCGGTGGTGGGTCTACTAGTGACATCGAGCTCAC CCAG-3′611 scBstSpeSacR (SEQ ID NO: 27):3′-GTGGTGCCAGTGGCAGAGGAGTCCGCCACCGCCTAGTCCACCGCCACCTTCACCGCCACCACCCAGATGATCACTGTAGCTCGAGTG GGTC-5′527 Reverse (SEQ ID NO: 28): 5′-CAGGAAACAGCTATGAC-3′619 E8VHf-SfiNcoPstR (SEQ ID NO: 29):3′-GACGCCGGGTCGGCCGGTACCGGCTCCAAGTCGACGTCGTCA-5′Primers 610 and 611 were annealed together, and then cloned intopFCAH3-E8T at the BstPI-SacI site. Single chain preparation was carriedout based on this construct. Further, PCR was carried out using primers527 and 619, and the resulting products were inserted into the constructat the HindIII-PstI site to introduce SfiI and NcoI sites.

Example 2 Preparation of an Immunoglobulin Light Chain Library

2-1 Isolation of Immunoglobulin Light Chain Genes Using PCR

2.6 μg mRNA was extracted from bone marrow cells (specimen No. 59) 4×10⁷cells, umbilical blood lymphocytes or peripheral blood lymphocytes usinga commercially available kit (QuickPrep Micro mRNA Purification Kit;Pharmacia Biotech). cDNA was prepared from the mRNA. cDNA preparationwas carried out with the SuperScript Preamplification System fromGibcoBRL. The primer used was oligo dT. PCR was carried out using theobtained cDNA as a template and 5′ primer (κ1 to κ6, κ1 to κ6) and 3′primer (hCKASC primer or hCLASC primer) for the light chain gene. Afterbeing treated with phenol, the PCR products were ethanol-precipitated.The DNA was suspended in 10 μl of TE buffer. The nucleotide sequences ofthe primers and PCR condition used are as follows. The underlines in thenucleotide sequence of the primers for the light chain gene indicateSfiI or AscI site.

5′-primer κ1 to κ6 hVK1a (SEQ ID NO: 30):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GACATCCAGA TGACCCAGTCTCChVK2a (SEQ ID NO: 31): GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GATGTTGTGATGACTCAGTCTCC hVK3a (SEQ ID NO: 32):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GAAATTGTGT TGACGCAGTCTCChVK4a(SEQ ID NO: 33): GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GACATCGTGATGACCCAGTCTCC hVK5a (SEQ ID NO: 34):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GAAACGACAC TCACGCAGTCTCChVK6a (SEQ ID NO: 35): GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GAAATTGTGCTGACTCAGTCTCC 5′-primer λ1 to λ6 hVL1 (SEQ ID NO: 36):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGTCTGTGT TGACGCAGCCGCChVL2 (SEQ ID NO: 37): GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGTCTGCCCTGACTCAGCCTGC hVK3a (SEQ ID NO: 38):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC TCCTATGTGC TGACTCAGCCACChVL3b (SEQ ID NO: 39): GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC TCTTCTGAGCTGACTCAGGACCC hVL4 (SEQ ID NO: 40):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CACGTTATAC TGACTCAACCGCChVL5(SEQ ID NO: 41): GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGCTGTGCTCACTCAGCCGCC hVL6 (SEQ ID NO: 42):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC AATTTTATGC TGACTCAGCCCCA 3′primer hCKASC (SEQ ID NO: 43):TCGACTGGCGCGCCGAACACTCTCCCCTGTTGAAGCTCTTTGTG 3′primer HCLASC (SEQ ID NO: 44):TCGACTGGCGCGCCGAACATTCTGTAGGGGCCACTGTCTTCTCPCR Condition

cDNA 2 μl 10x buffer #1 (attached to KOD) 10 μl  dNTP mix (2.0 mM) 10μl  25 mM MgCl₂ 4 μl 5′ primer (100 pmol/μl) 1 μl 3′ primer (100pmol/μl) 1 μl sterilized MilliQ 71 μl  KOD DNA polymerase (Toyobo 2.5U/μl) 1 μl

35 cycles of: 94° C., 1 minute; 55° C., 2 minutes; 74° C., 1 minute.

2-2 A Method for Preparing a Light Chain Gene Library by Selecting LightChains Suitable for Preparing the Library

2-2-1 Insertion of the Light Chain Gene into a Phagemid

The PCR product obtained in Example 1 was treated with restrictionenzymes under the following condition:

PCR product 10 μl  10x NEB4 (attached to AscI) 5 μl 10x BSA (attached toSfiI) 5 μl sterilized MilliQ 28 μl  AscI (10 U/μl; NEW ENGLAND BiolabsInc.) 1 μl SfiI (20 U/μl; NEW ENGLAND Biolabs Inc.) 1 μl

The reaction mixture was incubated at 37° C. for one hour and then at50° C. for one hour. After reaction, a 10-μl aliquot of the mixture waselectrophoresed in an agarose gel. A band of approximately 600 bp wascut off and the DNA was purified using a Gene Clean II Kit (Funakoshi).pFCAH9-E8d (FIG. 2) was treated with the same restriction enzymes asused to digest the PCR products, and then purified using a Gene Clean IIKit. The vector was ligated with the restriction enzyme-treated PCRproducts by incubating at 16° C. for a period from 4 hours to overnightunder the following condition.

pFCAH9-E8d treated with restriction enzyme 2 μl PCR product treated withrestriction enzyme 1 μl 10x ligation buffer 1.5 μl   (attached to T4 DNAligase) 10 mM ATP 1.5 μl   sterilized MilliQ 8 μl T4 DNA ligase (10U/μl; Takara Shuzo) 1 μl2-2-2 Introduction of Phagemid into E. coli

E. coli DH12S was transformed with the obtained DNA ligate as follows.Specifically, the DNA ligate was ethanol-precipitated once, and thendissolved in 3 μl of ⅕TE (which is TE diluted 5 times with sterilizedMilliQ). A 1.5-μl aliquot of the solution was combined with 20 μl of aliquid of competent cell DH12S (GIBCO BRL) and then the DNA waselectroporated into the E. coli cells under the following condition:

Electroporator Cell-Porator (BRL; Cat. series 1600) Settings; voltagebooster 4 kΩ capacitance 330 μF DC volts LowΩ charge rate Fast2-2-3 Secretion of an Fab-cp3-Type Antibody from E. coli CellsTransformed with Phagemid into Medium

The above-mentioned transformed E. coli cells were inoculated to 2 ml oftransformation medium (SOB). After the cells were cultured while beingshaken at 37° C. for one hour, an aliquot of the culture was plated onan agar medium (ampicillin plate). The remaining was cultured in 2×YTmedium containing 0.1% glucose and 100 μg/ml ampicillin, and then storedas a glycerin stock. The agar plate was incubated at 30° C., and thencolonies grown were picked up with toothpicks for isolation. Plasmidswere prepared from the colonies. The nucleotide sequences of the lightchain genes were determined using the plasmids.

SOB medium: the following components were added to 950 ml of purifiedwater; the mixture was shaken to dissolve them completely. Then, 10 mlof 250 mM KCl solution was added to the solution, and the pH of themixture was adjusted to 7.0 using 5N NaOH. The volume of the mixture wasadjusted to 1,000 ml by adding purified water thereto, and sterilized byautoclaving for 20 minutes. Immediately before use, 5 ml of sterilized2M MgCl₂ was added to the medium.

bacto-tryptone 20 g bacto-yeast extract  5 g NaCl 0.5 g 2×YT medium: the following components were added to 900 ml of purifiedwater, and the mixture was shaken to dissolve them completely. The pH ofthe mixture was adjusted to 7.0 using 5N NaOH. The volume of the mixturewas adjusted to 1,000 ml by adding purified water thereto. The mediumwas sterilized by autoclaving for 20 minutes.

bacto-tryptone 16 g bacto-yeast extract 10 g NaCl  5 gOther reagents were purchased from the following suppliers.

Supplier Name of item Sigma Ampicillin sodium salt Wako Pure ChemicalIndustries Phenol Sigma BSA DIFCO 2× YT medium Wako Pure ChemicalIndustries Kanamycin sulfate Nacalai Tesque Polyethylene glycol 6000Nacalai Tesque Tween20 Katayama Chemical NaCl Wako Pure ChemicalIndustries IPTG Wako Pure Chemical Industries Skimmed milk Wako PureChemical Industries Sodium azide Wako Pure Chemical IndustriesTriethylamine Wako Pure Chemical Industries Hydrogen peroxide Wako PureChemical Industries OPD tablet Wako Pure Chemical Industries ethanol

The above-mentioned treatment was practiced for all of κ1, κ2, κ3, κ4,κ5, and κ6, and λ1, λ2, λ3a, λ3b, λ4, λ5, λ6, λ7, λ8, λ9, and λ10 toconfirm whether clones of interest were isolated. Then, clones from eachgroup such as κ1 and κ2 were combined together so that the combiningratio could mimic the in vivo distribution. It has been known how theexpression levels are actually distributed in vivo with respect to genesof the respective light chain groups. To prepare a VL library, thesegene clones amplified by PCR and inserted into the vector were combinedtogether so that the combining ratio could mimic the in vivodistribution. The component ratio of the respective families in the VLlibrary is shown below.

TABLE 2 Component Component In vivo ratio in the ratio in distributionVL library KL200 Family (%)* (%) (%) Vκ1 39 37 30.7 Vκ2 12 12 19.8 Vκ336 35 33.7 Vκ4 12 12 10.9 Vκ5  1  2 5.0 Vκ6 —**   2*** 0.0 *Griffith A Det al. EMBO J. (1994) 13, 3245-60. **No description in the report***Mixture containing equal amounts of cDNA prepared with primer VK6-2and cDNA prepared with primer VK6-3

TABLE 3 Component Component In vivo ratio in the ratio in distributionVL library KL200 Family (%)* (%) (%) Vλ1 43  41  34.1 Vλ2 15   15*³ 15.2Vλ3 34   32*⁴ 25.3 Vλ4 0 1.5*⁵ 0.0 Vλ5 0 1.0*⁶ 11.1 Vλ6 0 1.0   14.1 Vλ76 6   0.0 Vλ8 1 1   0.0 Vλ9 1 1   0.0  Vλ10 —*² 1   0.0 *Griffith A D etal. EMBO J. (1994) 13, 3245-60. *²No description in the report *³Mixturecontaining 5% of cDNA prepared with primer VL2 and 10% of cDNA preparedwith primer VL2-2 *⁴Mixture containing 17% of cDNA prepared with primerVL3a-2 and 15% of cDNA prepared with primer VL3b *⁵Mixture containing0.5% of cDNA prepared with primer VL4a, 0.5% of cDNA prepared withprimer VL4b, and 0.5% of cDNA prepared with primer VL4c *⁶Mixturecontaining 0.5% of cDNA prepared with primer VL5abde and 0.5% of cDNAprepared with primer VL5c

Then, sequencing was carried out to confirm the nucleotide sequences ofapproximately 1,000 types of light chain genes selected at random fromthe VL library. Specifically, the nucleotide sequences were determinedby the dideoxy method using a fluorescent primer huCH1J(5′-ATTAATAAGAGCTATCCCGG-3′/SEQ ID NO: 45) and a thermo sequence kit(Amersham Pharmacia) in the automatic sequencer L1-COR4200L(S)-2(Aloka). Redundant clones were removed after the determined nucleotidesequences were compared. Further, clones which had been confirmed tohave no deletion as compared with the data in DNA databases werecombined with a clone for the heavy chain gene, VH3-4, whose expressionhad previously been confirmed. With such combinations, the expressionwas studied experimentally. The procedure used is described below. Theamino acid sequence of VH3-4 is shown in SEQ ID NO: 1.

First, VH3-4 was double-digested with HindIII and XhoI to obtain theheavy chain gene, and then purified with a Gene Clean II Kit. On theother hand, clones of light chain genes, which had been confirmed tohave no deletion, were also double-digested with HindIII and XhoI toobtain the light chain genes, and then purified using a Gene Clean IIKit. The fragments were ligated with the VH3-4 heavy chain gene toprepare a series of combinations of the genes. E. coli DH12S wastransformed with the obtained DNA ligate. The colonies grown wereinoculated to media in test tubes, and the expression was induced byadding IPTG thereto. Thus, the Fab-cp3-type antibodies were expressedand secreted into culture supernatants. Even without infecting helperphage, the Fab-cp3-type antibodies were expressed and secreted intoculture supernatants, when the culture was continued for about 20 hours.ELISA was carried out using these culture supernatants by the followingprocedure.

2-2-4 ELISA Test for the Exact Expression and Association of the HeavyChain and Light Chain

(1) Preparation of 96-Well Microtiter Plates on which an Antibody hasbeen Immobilized

A solution of anti-κ antibody (MBL; Code No. 159) was diluted to 1.25μg/ml with 0.01 M sodium phosphate buffer (pH8.0) containing 0.1% NaN₃,and 100-μl aliquots of the solution were added to a microtiter plate.The anti-κ antibody was immobilized (adsorbed) on each well by allowingthe plate to stand still at 4° C. overnight. The reaction solution wasdiscarded, and 200 μl of 0.01 M sodium phosphate buffer (pH8.0)containing 5% BSA and 0.1% NaN₃ was added to each well of the microtiterplate. To prevent non-specific adsorption, the plate was subjected toblocking treatment, which was carried out by allowing the plate to standstill at 37° C. for two hours.

Then, an anti-λ antibody (MBL code No. 159), whose non-specificreactivities had been blocked by absorption, was diluted to 2.5 μg/mlwith 0.0.1 M sodium phosphate buffer (pH8.0) containing 0.1% NaN₃, and100-μl aliquots were added to the microtiter plate. The plate wasallowed to stand still in a cold room overnight. The reaction solutionwas discarded, and 200 μl of 0.01 M sodium phosphate buffer (pH8.0)containing 5% BSA and 0.1% NaN₃ was added to each well of the microtiterplate. To prevent non-specific adsorption, the plate was subjected toblocking treatment, which was carried out by allowing the plate to standstill at 37° C. for two hours.

(2) Primary Reaction

100 μl each of a human Fab solution (10 μg/ml) as a positive control andPBS/0.1% NaN₃ as, a negative control was added to a microtiter plate.The expression of Fab-cp3-type antibody was induced by adding IPTG.100-μl aliquots of the original culture supernatants were added to themicrotiter plate, and the plate was incubated at 37° C. for one hour.

(3) Secondary Reaction

The microtiter plate after the primary reaction was washed five timeswith 0.05% Tween20-PBS. Then, 100-μl aliquots of an anti-Fd antibodysolution diluted to 1 μg/ml with PBS/0.1% NaN₃ were added to themicrotiter plate. The plate was incubated at 37° C. for one hour.

(4) Tertiary Reaction

The microtiter plate after the secondary reaction was washed five timeswith 0.05% Tween20-PBS. Then, 100-μl aliquots of alkalinephosphatase-conjugated anti-sheep IgG antibody diluted with PBS/0.1%NaN₃ (4000-fold dilution) were added to the microtiter plate. The platewas incubated at 37° C. for one hour.

(5) Color Development and Spectrometry

The microtiter plate after the tertiary reaction was washed five timeswith 0.05% Tween20-PBS. Then, 100-μl aliquots of a coloring substratesolution (SIGMA 1040; a phosphatase substrate tablet of SIGMA 10401 wasdissolved in 5 ml of 50 mM diethanol amine (PH9.8)) were added to themicrotiter plate. The plate was incubated at room temperature. When theabsorbance at 405 nm reached 0.5 or more, a stop solution was added tothe plate. The absorbance was determined by spectrometry in a plateleader (Titertek Multiscan MCC).

Clones assessed as positive (the absorbance was 0.5 or more) by ELISAwere assumed to successfully express the Fab-cp3-type antibody and hold.Then, from such clones, 100 clones having higher reactivity wereselected for each of κ chain gene and λ chain gene. The two sets ofclones were combined together to prepare the library KL200 which was acollection of clones successfully expressing the Fab-cp3-type antibodyand holding.

Example 3 Preparation of a Combinatorial Library Comprising Light ChainGene Library and Heavy Chain Gene Library

3-1-1 Isolation of Immunoglobulin Heavy Chain Genes by PCR

cDNA was prepared from lymphocytes of umbilical blood, bone marrowfluid, and peripheral blood, and tonsil by the same procedure as used inExample 2-1 using human R primer (primer 634 indicated below) or randomhexamer. PCR was carried out using the cDNA as a template and 5′ primers(VH1 to VH7) and a 3′ primer (a mixture containing equal amounts of fourtypes of human JH primers; primers 697 to 700 indicated below), or humanμ primer (primer 634 indicated below), which had been designed to clonehuman antibody heavy chain genes; the primers are listed below. In theTable, SfiI sites are underlined. Since hVH2a does not belong to thegerm line VH2 family, VH2a-2 primer was newly designed. In addition,since hVH4a does not correspond to all the VH4 family, hVH4a-2 primerwas newly designed. Further, since VH5a does not correspond to the germline VH5 subfamily, VH5a-2 primer was newly designed. A new primer hVH7was designed for VH7. These genes were also amplified, and inserted intopscFvCA-E8VHd(0-2). The nucleotide sequences were determined to confirmthe structures of amplified genes. The sequence of hVH5a-2 was highlyhomologous to that of hVHla, and thus the gene product was predicted tobe similar to that from the PCR product amplified with hVH1a; therefore,hVH5a-2 was not used. After being phenol-treated, the PCR products wereethanol-precipitated and suspended in 10 μl of TE buffer.

634 humμCH1R (SEQ ID NO: 46): ATGGAGTCGGGAAGGAAGTC

Primes Used for Amplifying Genes of Each VH Family

Human VH Primer (SfiI Sites are Underlined)

628 hVH1a (SEQ ID NO: 47): GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGTGCAGCTGGTGCAGTCTGG 629 hVH2a (SEQ ID NO: 48):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGTCAAC TTAAGGGAGTCTGG630 hVH3a (SEQ ID NO: 49): GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GAGGTGCAGCTGGTGGAGTCTGG 631 hVH4a (SEQ ID NO: 50):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGTGCAG CTGCAGGAGTCGGG632 hVH5a (SEQ ID NO: 51): GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGTGCAGCTGTTGCAGTCTGC 633 hVH6a (SEQ ID NO: 52):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGTACAG CTGCAGCAGTCAGG629-2 hVH2a-2 (SEQ ID NO: 53):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGRTCACC TTGAAGGAGTCTGGTCC631-2 hVH4a-2 (SEQ ID NO: 54):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGTGCAG CTACAGCAGTGGGG632-2 hVH5a-2 (SEQ ID NO: 55):GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC GAGGTGCAG CTGGTGCAGTCTGG712 hVH7 (SEQ ID NO: 56): GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC CAGGTGCAGCTGGTGCAATCTGGGTCTGAGT 697 hJH1-2 (SEQ ID NO: 57):GGTGGAGGCACTCGAGACGGTGACCAGGGTGC 698 hJH3 (SEQ ID NO: 58):GGTGGAGGCACTCGAGACGGTGACCATTGTCC 699 hJH4-5 (SEQ ID NO: 59):GGTGGAGGCACTCGAGACGGTGACCAGGGTTC 700 hJH6 (SEQ ID NO: 60):GGTGGAGGCACTCGAGACGGTGACCGTGGTCC Human JH primer (BstPI and XhoI sitesare underlined)

cDNA 2 μl 10× buffer #1 (attached to KOD) 10 μl  dNTP mix (2.0 mM) 10μl  25 mM MgCl₂ 4 μl 5′ primer (100 pmol/μl) 1 μl 3′ primer (100pmol/μl) 1 μl sterilized MilliQ 71 μl  KOD DNA polymerase (2.5 U/μl;Toyobo) 1 μlPCR condition: 35 cycles of: 94° C., 1 minute; 55° C., 2 minutes; 74°C., 1 minute.3-1-2 Preparation of Heavy Chain Gene Library

The PCR products obtained in 3-1-1 were treated with restriction enzymesunder the following condition:

PCR product 10 μl  10× K buffer (Takara Shuzo) 5 μl sterilized MilliQ 33μl  HindIII (15 U/μl; Takara Shuzo) 1 μl XhoI (12 U/μl; Takara Shuzo) 1μl

The reaction mixture was incubated at 37° C. for two hours. Afterreaction, a 10-μl aliquot of the mixture was electrophoresed in anagarose gel. A band of approximately 400 bp was cut off and the DNA waspurified using a Gene Clean II Kit (Funakoshi). pscFvCA-E8VHd (FIG. 2)were treated with the same, restriction enzymes as used to digest thePCR products, and then purified using a Gene Clean II Kit. The vectorwas ligated with the restriction enzyme-treated PCR products byincubating at 16° C. for a period from 4 hours to overnight under thefollowing condition.

Restriction enzyme-treated pscFvCA-E8VHd 2 μl Restriction enzyme-treatedPCR product 1 μl 10× ligation buffer 1.5 μl   (attached to T4 DNAligase) 10 mM ATP 1.5 μl   sterilized MilliQ 8 μl T4 DNA ligase (10U/μl; Takara Shuzo) 1 μl3-1-3 Introduction of Phagemid into E. coli

E. coli DH12S was transformed with the obtained DNA ligate.Specifically, the DNA was ethanol-precipitated once, and then dissolvedin 3 μl of ⅕TE (which is TE diluted 5 times with sterilized MilliQ). A1.5-μl aliquot of the solution was suspended in 20 μl of a solution ofcompetent cell DH12S (GIBCO BRL), and then the DNA was transformed intothe E. coli cells by electroporation.

Electroporator Cell-Porator (BRL; Cat. series 1600) Settings; voltagebooster 4 kΩ capacitance 330 μF DC volts LowΩ charge rate Fast

The E. coli cells transformed by the above-mentioned procedure wereinoculated to 2 ml of transformation medium (SOB). After the cells werecultured while being shaken at 37° C. for one hour, an aliquot of theculture was plated on an agar medium (ampicillin plate). The remainingwas cultured in 2×YT medium containing 0.1% glucose and 100 μg/mlampicillin, and then stored as a glycerin stock. The agar plate wasincubated at 30° C., and then colonies grown were picked up withtoothpicks for isolation. Plasmids were prepared from the colonies, andthe nucleotide sequences of the heavy chain genes were determined usingthe plasmids. The above-mentioned treatment was practiced for all of VH1to VH7 to confirm whether clones of interest were isolated. Then, toprepare a VH library, clones from each group (family) were combinedtogether so that the combining ratio could mimic the in vivodistribution. The component ratio of the respective families in the VHlibrary is shown below.

TABLE 4 Component In vivo ratio in the distribution VH library Family(%)* (%) VH1 25  29** VH2 6.6 7 VH3 40 40  VH4 19  19*** VH5 5 —** VH63.8 4 VH7 1.2 2 *Griffith A D et al. EMBO J. (1994) 13, 3245-60.**Actually, VH1 and VH5 are inseparable in tabulation, because both areamplified with identical primers. ***A mixture was prepared by combiningcDNA synthesized with primer VH4 and cDNA synthesized with primer VH4-2at this ratio.3-2 Preparation of a Combinatorial Gene Library

200 μg of the VH library was double-digested with HindIII and XhoI underthe condition as described below to obtain the heavy chain gene, and thedigest was purified with a Gene Clean II Kit.

VH library 200 μg 100 μl 10× K buffer (Takara Shuzo) 40 μL sterilizedMilliQ 205 μl HindIII (40 U/μl; Takara Shuzo) 30 μL XhoI (50 U/μl;Takara Shuzo) 25 μl

The light chain gene clone KL200, which had been confirmed to have nodeletion, and the vector pFCAH9-E8d in which the VL library had beeninserted, were also double-digested with HindIII and XhoI under thefollowing condition. Then, the fragments containing the light chain genewere purified using a Gene Clean II Kit.

pFCAH9-E8d containing KL200 or VL library 100 μl  as an insert 100 μg10× K buffer (Takara Shuzo) 40 μl sterilized MilliQ 230 μl  HindIII (40U/μl; Takara Shuzo) 15 μl XhoI (50 U/μl; Takara Shuzo) 15 μl

Then, the fragments of the VH gene library and pFCAH9-E8d vector inwhich the light chain gene had been inserted were ligated together underthe following condition at 16° C. overnight.

Restriction enzyme-treated 50 μl Fragments of the VH library 10 μgpFCAH9-E8d containing 50 μl Restriction enzyme-treated KL200 orFragments of the VL library 40 μg 10× ligation buffer 100 μl (attachedto the T4 DNA ligase) 10 mM ATP 100 μL Sterilized MilliQ 670 μl T4 DNAligase (10 U/μl; Takara Shuzo) 30 μl

E. coli DH12S was transformed with the DNA after the reaction.Specifically, the DNA was ethanol-precipitated once, and then dissolvedin 30 μl of ⅕TE (which is TE diluted 5 times with sterilized MilliQ).The DNA solution was combined with 500 μl of competent cell DH12S (GIBCOBRL), and then electroporation was carried out.

Electroporator Cell-Porator (BRL; Cat. series 1600) Settings; voltagebooster 4 kΩ capacitance 330 μF DC volts LowΩ charge rate Fast

The E. coli cells after the above-mentioned treatment were inoculated to12 ml of transformation medium (SOB). After the cells were culturedwhile being shaken at 37° C. for one hour, an aliquot of the culture wasplated on an agar medium (ampicillin plate). The remaining was culturedin 500 ml of 2×YT medium containing 0.1% glucose and 100 μg/mlampicillin, and then stored as a glycerin stock. The agar plate wasincubated at 30° C., and then the number of clones obtained wasestimated based on the number of colonies grown. 5×10¹⁰ clones wereobtained for each library.

cDNAs of each VH family, which had been synthesized from tonsil mRNAusing random hexamer as a primer, were cloned into pscFvCA-E8VHd vector.The cDNA constructs were combined with KL200 to prepare a combinatoriallibrary AIMS1 (the number of independent clones was 1.28×10¹⁰).

cDNAs of each VH family, which had been synthesized from mRNAs fromumbilical blood, bone marrow fluid, peripheral blood, and tonsil usingthe human m primer, were cloned into pscFvCA-E8VHd vector. The cDNAconstructs were combined with KL200 to prepare a combinatorial genelibrary AIMS2 (the number of independent clones was 3.20×10¹⁰).

The library of VH family cDNAs, which had been synthesized from mRNAsprepared from umbilical blood, bone marrow fluid, peripheral blood, andtonsil using the human m primer, was combined with the VL library toprepare a combinatorial library AIMS3 (the number of independent cloneswas 4.50×10¹⁰).

Another combinatorial library was prepared by combining the libraries atthe following ratio:

(AIMS1+AIMS2):AIMS3=1:1. The resulting phage antibody library was namedAIMS4 and contained 1×10¹¹ independent clones.

3-3 Preparation of Phage Libraries Using the Combinatorial GeneLibraries

3-3-1 Preparation of Phage Libraries

2.5 ml of an AIMS4 suspension was added to 300 ml of 2×YT mediumcontaining 1% glucose and 100 μg/ml ampicillin in a 5-liter flask.Culture was continued at 37° C. while the flask was being shaken. Theabsorbance at wavelength 600 nm was measured at 1-hour intervals. Theincubation was continued until the absorbance reached 1.0. 12 ml/flaskof helper phage liquid (M13KO7) was added to the culture medium toinfect the helper phage. Culture was continued at 37° C. for 2 hours.Thus, helper phage-infected DH12S cells were prepared.

600 ml of 2×YT medium, 1.2 ml of 100 μg/ml ampicillin, 0.8 ml of 50μg/ml kanamycin, and 200 ml of the helper phage-infected DH12Ssuspension were added to twenty four 3-liter flasks. The flasks wereincubated at 37° C. while being shaken. The absorbance at wavelength 600nm was measured at 2-hour intervals. Ampicillin was freshly added at afinal concentration of 200 μg/ml to each flask at every absorbancemeasurement. Incubation was continued until the absorbance at wavelength600 nm reached 1.3.

The bacterial cultures were centrifuged at 8,000 rpm for ten minutes at4° C., and the supernatants were collected. 4 liters of 20% polyethyleneglycol/2.5M NaCl was added to each supernatant. The mixture was stirredgently for about 20 minutes, and then centrifuged at 8,000 rpm for 20minutes at 4° C. The resulting precipitate was dissolved in 1 liter ofPBS. 200 ml of 20% polyethylene glycol/2.5M NaCl was added to thesolution and the mixture was stirred gently for about 20 minutes. Then,the mixture was centrifuged at 8,000 rpm for 20 minutes at 4° C. Thesupernatant was discarded, and the remaining material was furthercentrifuged at 8,000 rpm for three minutes at 4° C. The pellet wascollected. The precipitate was dissolved in PBS containing 0.05% NaN₃.The solution was centrifuged at 1,000 rpm for 15 minutes at 4° C., andthen the supernatant was collected. The supernatant was againcentrifuged at 8,000 rpm for three minutes at 4° C. The resultingsupernatant was collected.

The titer of recovered phage liquid was tested as described below.Specifically, the phage liquid was diluted 10⁶ times, 10⁷ times, or 10⁸times with PBS. A 10-μL aliquot of the dilute was combined with 990 μlof DH12S for infection. The mixture was incubated at 37° C. for onehour. A 100-μl aliquot of the culture was plated on an LBGA plate. Theplate was incubated at 30° C. for 18 hours. The phage titer of originalliquid before dilution was estimated based on the number of colonies.The phage stock solution was diluted with PBS containing 2% skimmed milkand 0.05% NaN₃ to 2×10¹⁴ particles/ml.

3-3-2 A Method for Enriching Phage Particles Expressing Fab-cp3 on theSurface

The library prepared as described above was designed so that phageparticles expressing Fab-cp3 on the surface could be selectivelyenriched and the level of contamination of helper phage particles andphage particles expressing no Fab-cp3 would be reduced. Specifically,His6 peptide (SEQ ID NO: 106) (histidine tag) was attached to theC-termini of heavy chains expressed on the phages constituting theabove-mentioned library. Phage particles expressing the histidine tagcan be recovered simply by trapping them on nickel ion or the like.Specifically, gel containing nickel ions (Ni-NTA agarose, etc) can beused. The procedure used was as follows.

For blocking, Ni-NTA agarose was incubated in PBS containing 2% skimmedmilk and 0.1% Tween-20 (hereinafter referred to as blocking buffer) atroom temperature for 30 minutes. Then, in the blocking buffer, phagesexpressing, on the surface, Fab comprising the heavy chain withoutHis-Tag (pFCA-E9HL4; phage His-) and phages expressing, on the surface,Fab comprising the heavy chain with His-Tag (pFCAH6-D1.3HLφ; phage His+)were combined together at a ratio of: phage His−:phage His+=100:1. 250μl of the phage solution (1×10¹⁰ CFU in total) was combined with Ni-NTAagarose, and the mixture was incubated at room temperature for one hour.The Ni-NTA agarose was washed with the blocking buffer, and then 500 μlof 0.5 M imidazole (pH 7.55) was added thereto to elute the phageparticles bound to the Ni-NTA agarose.

The eluted phage particles were recovered according to the method asdescribed below (Example 4-3). Then, the recovered clones were examined.Of 23 clones, 15 were phages of His+ (Table 3). This suggests thatNi-NTA agarose enriched His6 peptide-containing phage particles 53 times(6His peptide disclosed as SEQ ID NO: 106).

The findings described in (4) demonstrate that this treatment canimprove the library performance or increase screening efficiency.

TABLE 5 Phage Phage without carrying His6-tag His6-tag Before Ni-NTAagarose 100 1 treatment After Ni-NTA agarose 15 8 treatment ** 6xHis tagis disclosed as SEQ ID NO: 106

Example 4 Selection of Phages Binding to a Specific Antigen from a PhageLibrary (Part 1)

A procedure of selecting phages binding to a specific antigen from aphage library is herein referred as “screening”. Screening of theantibody library of the present invention was carried out using tetanustoxoid as an antigen.

4-1 Preparation of Test Tubes to be Used in the Screening

The concentration of the antigen (tetanus toxoid) was adjusted to 20μg/ml with PBS, and a 3-ml aliquot of the solution was added to each ofthree test tubes (Maxisorp; Nunc). The mixtures were incubated at 4° C.for 18 hours to absorb the antigen on the inner surface of the tubes.After adsorption, the antigen solution was discarded, and a 3-ml aliquotof a PBS solution containing 2% skimmed milk was added thereto. Blockingwas carried out by incubating the test tubes at 25° C. for one hour, toavoid non-specific adsorption of phage antibodies on the test tubes.

4-2 Screening Method

A 3-ml aliquot of an AIMS4 phage library (1×10¹⁴ CFU) in 9 ml of PBScontaining 2% skimmed milk and 0.1% Tween-20 was added to each of thethree antigen-immobilized test tubes. The tubes were incubated at 25° C.for 2 hours, and then washed 4 times with PBS containing 0.1% Tween-20,4 times with PBS, and once sterilized extra pure water (prepared byMilliQ).

Then, phage particles bound to the antigen-immobilized test tubes wererecovered by the procedure as described below. Specifically, 3 ml of 0.1M triethylamine (pH 12.3) was added to each test tube, and the tubeswere incubated while being rotated with a rotator at room temperaturefor 20 minutes to dissociate the phage particles from tube surface.Then, 1.1 ml of 1 M Tris-HCl buffer (pH6.8) was added to the tubes toneutralize the liquid. The neutralized phage solutions were collected.

4-3 Amplification of the Collected Phages

The phage solutions collected were treated as follows:

(a) the phage was infected to E. coli cells;

(b) helper phage was infected to E. coli cells; and

(c) the resulting phage particles were collected.

The phage particles in the solution were thus purified and amplified.

(1) Infection of Phage Particles to E. coli Cells

E. coli (DH12S) cells were cultured in 50 ml of 2×YT medium, until theabsorbance at 600 nm reached 0.5. Then, the phage solution prepared asdescribed above was added to the culture. Shaking culture was continuedat 37° C. for one hour.

(2) Infection of Helper Phage

A 62.3-ml aliquot of the culture solution obtained by (1) was added to425 ml of 2×YT medium containing 12.5 ml of 40% glucose and 0.5 ml of100 μg/ml ampicillin. Culture was continued at 37° C., until theabsorbance at 600 nm reached 0.5. Then, the culture liquid wascentrifuged at 5,000 rpm at 4° C. for ten minutes to precipitate thebacterial cells. The cells were collected and suspended in 300 ml of2×YT medium containing 0.3 ml of 100 mg/ml ampicillin. A 1/100 volume ofhelper phage M13K07 was added to the culture, and then shaking culturewas continued at 37° C. for one hour.

The culture solution was added to 900 ml of medium (2×YT mediumcontaining 100 μg/ml ampicillin and 70 μg/ml kanamycin) pre-warmed at37° C. The resulting culture solution was incubated at 37° C. overnight.

(3) Phage Recovery

The culture solution prepared in (2) was centrifuged at 7,000 rpm forten minutes at 4° C., and then a ⅕ volume of 20% polyethylene glycolcontaining 2.5 M sodium chloride was added to the resulting supernatant.The mixture was allowed to stand still at room temperature for 20minutes, and then centrifuged at 8,000 rpm for 15 minutes at 4° C. Thepellet was collected, and sterilized PBS was added to the pellet in anamount that corresponded to 1/10 of the volume of the culture solution,thereby dissolving the pellet. Again, a ⅕ volume of a solution of 20%polyethylene glycol containing 2.5 M sodium chloride was added to thesuspension. The mixture was centrifuged at 10,000 rpm for 20 minutes at4° C., and then the supernatant was discarded. The sample was furthercentrifuged at 10,000 rpm for 2 minutes at 4° C. to remove residualliquid. PBS containing 0.05% NaN₃, which corresponded to 1/100 volume ofthe culture solution, was added to the pellet to suspend the phageparticles. Thus, the phage particles were collected.

4-4 Re-Screening Using the Amplified Phage

Screening was repeated using the amplified phage particles andantigen-immobilized test tubes by the same procedure as used in Example4-2. The washing step in the screening was important to dissociate thenon-specifically adsorbed phage particles and to select phages havinghigh affinity. Thus, the washing in the secondary and subsequentscreening was carried out under the following condition.

Secondary screening: 6 times with PBS containing 0.1% Tween-20, 6 timeswith PBS, once with sterilized extra pure water.

Tertiary screening: 13 times with PBS containing 0.1% Tween-20, 13 timeswith PBS, once with sterilized extra pure water.

4-5 Method for Evaluating Phage Screening

When the ratio of (the total number of phase particles recovered fromthe antigen-immobilized test tube)/(the total number of phase particlesin an antigen-immobilized test tube) becomes obviously smaller than thatof the previous screening in screenings conducted serially according tothe method described in Example 4-4, phage particles displaying thedesired antibody are estimated to be enriched. The number of phageparticles in the phage solution was determined by the followingprocedure.

(1) A serial dilution of phage particles was prepared as follows:

[1] 1×10⁻² dilution: 10 μl of the phage solution+990 μl of PBS

[2] 1×10⁻⁴ dilution: 10 μl of the dilute in [1]+990 μl of PBS

[3] 1×10⁻⁶ dilution: 10 μl of the dilute in [2]+990 μl of PBS

[4] 1×10⁻⁸ dilution: 10 μl of the dilute in [3]+990 μl of PBS

[5] 1×10⁻⁹ dilution: 100 μl of the dilute in [4]+900 μl of PBS

[6] 1×10⁻¹⁰ dilution: 100 μl of the dilute in [5]+900 μl of PBS

990 μl of DH12S was combined with each of 10-μl aliquots of the dilutesprepared in [4], [5], and [6] in the serial dilution. The mixtures wereincubated at 37° C. for one hour for infection, and 100-μl aliquotsthereof were plated on LBGA plates, followed by incubation at 30° C. for18 to 24 hours. The resulting colonies grown were counted. Normally, inthe above-described serial dilution, the dilute prepared in [4] provides50 or more colonies on a plate. The phage titer (the number of phageparticles) in 1 ml of the phage solution was estimated based on thenumber of colonies grown on the plate corresponding to the diluteprepared in [4] by the following formula.The number of phage particles in the phage stock solution=(the number ofcolonies/plate)×(1×10⁸)×10³ cfu/ml

The number of phage particles recovered was also estimated in the sameway, and thereby the number of phage particles displaying the antibodyagainst the antigen was determined for each screening. The result isshown in Table 6.

TABLE 6 (The number of phage particles The number The number The numberused)/ of The number of phage of phage (The number of screening ofwashing particles particles phage particles repetitions repetitions usedrecovered recovered) 1 4 1.15 × 10¹⁴ 6.1 × 10⁸ 1.9 × 10⁵ 2 6  3.9 × 10¹³2.2 × 10⁷ 1.8 × 10⁶ 3 8  1.2 × 10¹³ 5.4 × 10⁸ 2.2 × 10⁴4-6 Determination of Antigen-Binding Activity (Affinity) of AntibodiesObtained Through Screening

The antigen-binding activity (affinity) was determined for theantibodies selected by the screening as described above. Not phageantibodies but Fab-cp3-type antibodies were used as samples fordetermining the affinity. The assay method used was ELISA using 96-wellmicrotiter plates. The method for inducing the expression ofFab-cp3-type antibodies is described below in detail in Example 4-7.

First, ELISA plates were prepared as follows. 100 μl of 20 μg/ml antigenwas added to each well of 96-well microtiter plates (Maxisorp; Nunc),and then the plates were incubated at 4° C. for 18 hours forimmobilization. 200 μl of 5% BSA (blocking solution) was added to eachwell, and then the plates were incubated at 37° C. for one hour forblocking. After the blocking solution was discarded, the plates werewashed once with PBS. These plates were used for determining theaffinity. 100 μl of the culture supernatant recovered by the procedureas described in Example 4-7 was added to each well. The plates wereincubated at 25° C. for one hour. After incubation, the plates werewashed four times with PBS. 100 μl of peroxidase-conjugated anti-humanIgG (Medical and Biological Laboratories Co.), which had been diluted5,000 times, was added to the plates. The plates were incubated at 25°C. for one hour, and then washed again four times with PBS. 100 μl of asolution containing ortho-phenylenediamine and hydrogen peroxide wasadded to the plates. After the plates were incubated for a desired time,100 μl of 1.5 N phosphoric acid was added thereto to stop the reaction.The absorbance measured at a wavelength of 492 nm. Thus, of the 96clones, 77 clones were confirmed to have the activity (FIG. 10).

Further, it was tested whether 37 clones selected from the 77 clones hadthe neutralizing activity or not. Clones marked with circle in Table 7were tested for the neutralizing activity. At the time of testing theneutralizing activity, the nucleotide sequences of the clones had notyet been determined; some of the clones might share identical DNAsequences. The nucleotide sequences of the clones were determined andcompared to one another. Thus, clones sharing an identical nucleotidesequence are categorized into an identical group in Table 7. By thecomparison carried out after sequence determination, it was confirmedthat clones sharing an identical DNA sequence showed equivalent level ofaffinity determined by ELISA to each other.

4-7 Induction of Expression of Fab-cp3-Type Antibodies

Phage-infected E. coli cells were cultured in 2×YT medium containing 1%glucose and 100 μg/ml ampicillin at 30° C. for 18 hours. Then, a 5-μlaliquot of the culture was added to 1.5 ml of 2×YT medium containing0.1% glucose and 100 μg/ml ampicillin. The mixture was incubated at 30°C. for 4 hours. After the culture is completed, the absorbance at 600nm, which corresponded to E. coli cell density, was about 0.5.

IPTG (isopropyl-1-thio-β-D-galactoside) was added at a finalconcentration of 1 mM to the culture. Incubation was continued at 30° C.for 18 hours. A 1.5-ml aliquot of the culture was added to an Eppendorftube, and centrifuged at 10,000 rpm for 5 minutes at 4° C. The resultingculture supernatant was collected and sodium azide was added thereto ata final concentration of 0.1%. The supernatant was used as a sample.

ELISA as described in Example 2-3 was used to determine whether theFab-cp3-type antibody was expressed or not. Clones confirmed to beexpressed were tested for deletion by agarose electrophoresis. Deletionwas found in 8 clones. All of these clones were confirmed to have noantigen-binding activity.

4-8 Single-Clone Separation and Phagemid Purification

E. coli clones were selected from the plates for single-colonyseparation, which had been prepared according to Example 4-5 “Method forevaluating phage screening”, and then cultured in LBGA at 30° C. for 18hours. Then, phagemids were purified using a DNA extractor (PI-50 FileNo. 50) purchased from Kurabo.

4-9 Identification of Monoclonal Antibodies

Sequencing was carried out to determine the nucleotide sequences of thegenes encoding anti-tetanus toxin antibodies selected from the antibodylibrary of the present invention by screening. The heavy chain and lightchain genes were sequenced using the primers listed below by the dideoxymethod with a thermo sequence kit (Amersham Pharmacia) and an automaticsequencer. L1-COR4200L (S)-2 (Aloka). The result is summarized in Table7, where clones sharing an identical nucleotide sequence are categorizedinto an identical group based on the determined nucleotide sequences. Inthis Table, when having a same combination of the number for the H chainand the number for the L chain, clones comprise an identical nucleotidesequence. As seen in Table 7, 36 types of distinct clones were isolated.

Sequencing primers for the heavy chain: fluorescent labeled T7 primer(Aloka)

Sequencing primers for the light chain: fluorescent labeled primerhuCH1J (SEQ ID NO: 45)

TABLE 7 H-chain DNA Germ H-L gene The conduct of ELISA sequence VH1 linecombination neutralization Clone No. Absorbance ID NO: classificationKL200 VL ID NO. test TETM18 0.7321 1 DP10 (VH1) L3BUM6 31 1 ◯ TETM400.7185 1 DP10 (VH1) L3BUM6 31 1 ◯ TETM42 0.6266 1 DP10 (VH1) L3BUM6 31 1◯ TETM51 1.1731 1 DP10 (VH1) L3BUM6 31 1 TETM53 1.2410 1 DP10 (VH1)L3BUM6 31 1 TETM57 1.1961 1 DP10 (VH1) L3BUM6 31 1 TETM61 1.1833 1 DP10(VH1) L3BUM6 31 1 TETM63 1.1912 1 DP10 (VH1) L3BUM6 31 1 TETM68 1.1971 1DP10 (VH1) L3BUM6 31 1 TETM70 1.1911 1 DP10 (VH1) L3BUM6 31 1 TETM721.2544 1 DP10 (VH1) L3BUM6 31 1 TETM76 1.1253 1 DP10 (VH1) L3BUM6 31 1TETM81 1.1559 1 DP10 (VH1) L3BUM6 31 1 TETM84 0.9991 1 DP10 (VH1) L3BUM631 1 TETM85 0.9737 1 DP10 (VH1) L3BUM6 31 1 TETM24 0.7613 1 DP10 (VH1)13b-10b, 31 12 ◯ 13b-6b TETM02 0.6460 2 DP10 (VH1) 13b-12b 31 4 ◯ TETM090.7987 2 DP10 (VH1) 13b-12b 31 4 ◯ TETM44 0.4468 2 DP10 (VH1) 13b-12b 314 TETM93 0.6824 2 DP10 (VH1) 13b-12b 31 4 TETM14 0.6707 2 DP10 (VH1)13b-10b, 31 3 ◯ 13b-6b TETM25 0.6402 2 DP10 (VH1) 13b-10b, 31 3 ◯ 13b-6bTETM47 0.5186 2 DP10 (VH1) 13b-10b, 31 3 13b-6b TETM48 0.5222 2 DP10(VH1) 13b-10b, 31 3 13b-6b TETM86 0.8435 2 DP10 (VH1) 13b-10b, 31 3 ◯13b-6b TETM89 0.9117 2 DP10 (VH1) 13b-10b, 31 3 13b-6b TETM49 1.2680 2DP10 (VH1) VL library 31 13 ◯ TETM64 1.3011 2 DP10 (VH1) VL library 3114 TETM16 0.6521 3 DP10 (VH1) 13b-12b 31 2 ◯ TETM22 0.7705 3 DP10 (VH1)13b-12b 31 2 ◯ TETM26 0.7478 3 DP10 (VH1) 13b-12b 31 2 ◯ TETM33 0.7425 3DP10 (VH1) 13b-12b 31 2 ◯ TETM43 0.6578 3 DP10 (VH1) 13b-12b 31 2 ◯TETM45 0.6392 3 DP10 (VH1) 13b-12b 31 2 ◯ TETM46 0.6056 3 DP10 (VH1)13b-12b 31 2 ◯ TETM55 1.2684 3 DP10 (VH1) 13b-12b 31 2 TETM15 1.9333 3DP10 (VH1) L3BUM4 31 15 ◯ TETM96 0.6639 4 DP10 (VH1) L3BUM6 31 16 ◯TETM73 1.0694 4 DP10 (VH1) 13b-10b, 31 17 13b-6b TETM80 1.1280 4 DP10(VH1) VL library 31 7 TETM83 0.9669 4 DP10 (VH1) VL library 31 7 TETM030.7537 4 DP10 (VH1) 13b-1b 31 18 ◯ TETM71 1.1197 4 DP10 (VH1) L3BUM1 3119 TETM19 0.8051 5 DP10 (VH1) L3BUM6 31 8 ◯ TETM92 0.9295 5 DP10 (VH1)L3BUM6 31 8 TETM12 0.7983 5 DP10 (VH1) 13b-12b 31 20 ◯ TETM30 0.6473 5DP10 (VH1) 13b-1b 31 21 ◯ TETM28 0.6622 5 DP10 (VH1) L3BUM1 31 22 ◯TETM37 0.6879 6 DP10 (VH1) L3BUM6 31 6 ◯ TETM52 1.1214 6 DP10 (VH1)L3BUM6 31 6 TETM54 1.2341 6 DP10 (VH1) L3BUM6 31 6 TETM66 1.0685 6 DP10(VH1) 13b-10b, 31 23 13b-6b TETM41 0.6138 6 DP10 (VH1) 13b-1b 31 24 ◯TETM06 0.8287 7 DP10 (VH1) L3BUM6 31 5 ◯ TETM60 1.5267 7 DP10 (VH1)L3BUM6 31 5 TETM62 1.2762 7 DP10 (VH1) L3BUM6 31 5 TETM82 1.2137 7 DP10(VH1) L3BUM6 31 5 TETM08 0.8469 8 DP10 (VH1) L3BUM6 31 9 ◯ TETM75 0.95488 DP10 (VH1) L3BUM6 31 9 TETM32 0.7325 9 DP10 (VH1) L3BUM6 31 25 ◯TETM94 0.8597 9 DP10 (VH1) 13b-12b 31 26 TETM31 0.7118 10 DP10 (VH1)L3BUM6 31 27 ◯ TETM74 1.1040 10 DP10 (VH1) VL library 31 28 TETM340.6607 11 DP10 (VH1) L3BUM6 31 10 ◯ TETM67 1.2017 11 DP10 (VH1) L3BUM631 10 TETM17 0.7207 12 DP10 (VH1) L3BUM6 31 11 ◯ TETM29 0.7073 12 DP10(VH1) L3BUM6 31 11 ◯ TETM01 0.7729 13 DP10 (VH1) VL library 31 29 ◯TETM13 0.6374 13 DP10 (VH1) 13b-10b, 31 30 ◯ 13b-6b TETM21 0.7280 14DP10 (VH1) L3BUM6 31 31 ◯ TETM56 1.3910 15 DP10 (VH1) VL library 31 32TETM58 1.3259 16 DP77 (VH3) k6-17b DPK15 33 TETM69 0.9722 17 DP10 (VH1)L3BUM6 31 34 TETM88 2.1405 18 DP14 (VH1) VL library DPK9 35 ◯ TETM911.2441 19 DP75 (VH1) k1-5.b DPK9 364-10 Preparation of Fab-cp3-Type Antibodies to be Used for Testing theNeutralizing Activity

It was examined whether the antibodies selected from the antibodylibrary of the present invention by screening had the activity ofneutralizing tetanus toxin. First, each of the 77 clones exhibitingactivity as antibodies was pre-cultured before the antibody expressionwas induced. The bacterial cells preculture was combined with 2×YTmedium (YTA) containing 1% glucose and 100 mg/ml ampicillin, and thenincubated at 30° C. overnight. After the cells were further cultured in0.1% glucose-YTA at 30° C. for 3 hours, 1M IPTG was added thereto.Culture was continued at 30° C. for 20 hours. The culture solution wascentrifuged at 10,000 rpm for 5 minutes. The culture supernatant wascollected and ammonium sulfate was added thereto at a finalconcentration of 60%. The mixture was agitated for one hour, and thencentrifuged at 12,000 rpm for ten minutes. The resulting supernatant wasdiscarded, and the precipitate was dissolved with PBS. The resultingsolution was dialyzed against a 50 or more volume of PBS for 2 hours.This step was repeated three times. Then, the dialysate was transferredinto an Eppendorf tube, followed by centrifugation of the tube at 15,000rpm for 10 minutes at 4° C. After being filtered, the resultingsupernatant was tested for the activity of neutralizing tetanus toxin.

4-11 Examination of the Neutralizing Activity

First, to evaluate the toxicity of antibody itself, 0.2 ml of theantibody solution prepared as described above was injected into thecaudal vein and peritoneal cavity of mice. As the result, the antibodysolution had no toxicity. In the subsequent experiments, one hour afterthe antibody was injected, 10 times as much dose of tetanus toxin as theminimal lethal dose (1 MLD or about 2 LD₅₀) for mouse was injectedsubcutaneously in an inside area of thigh of the hind legs of mice. Asthe result, partial neutralization (retarded onset of the symptomscaused by toxin; 4 to 7 days) was recognized in about half of the miceinjected for testing each antibody. Mice, which were subjected toinjection of a neutralizing antibody (standard tetanus antitoxin) orantibody without the neutralizing activity, showed tetanustoxin-specific paralysis, and then died in about two days.

Clones that exhibited partial neutralizing activity were: TETM1, TETM13,TETM26, and TETM96. Thus, the above-described findings demonstrate thatuseful antibodies having the neutralizing activity can be yielded byscreening the antibody library of the present invention.

4-12 Sequence Determination for the Antibody Variable Region

As described above, the present inventors succeeded in preparing phagesexpressing antibody variable domains binding to and neutralizing tetanustoxoid (toxin).

Additionally, phage clones exhibiting similar neutralizing activity buthaving distinct nucleotide sequences were also obtained successfully byrepeating the above-mentioned step. Specifically, clones of Nos. TETM13and TETK36 were estimated to have strong neutralizing activity. Thenucleotide sequences of the heavy chain and light chain of these phageclones, and the amino acid sequences encoded by the nucleotide sequencesare shown below under the following SEQ ID Nos:

Clone No. Heavy chain Light chain TETM13 (nucleotide sequence) 63 64TETM13 (amino acid sequence) 81 82 TETK36 (nucleotide sequence) 65 66TETK36 (amino acid sequence) 83 84

Example 5 Comparison of a Light Chain Gene Library without Selection andthe KL200 Library

Library performance was compared between the combinatorial library(AIMS3), which had been prepared by combining a light chain gene librarywithout selection with a heavy chain, and the combinatorial library(AIMS1+AIMS2), which had been prepared by combining the light chain genelibrary KL200 with the heavy chain.

Firstly, the comparison showed that the antibody library AIMS1+AIMS2according to the present invention was overwhelmingly superior in thenumber of clones selected. Clones selected from the light chain genelibrary AIMS1+AIMS2, which had been subjected to the selection, are 29clones whose clone names are indicated in the column of “KL200” in Table7 shown above. On the other hand, 7 clones were selected from AIMS3(clones indicated by the label “VL library” in the column of “KL200” inTable 7). Namely, 80% or more (29/36) of the clones selected werederived from the library of the present invention, although thecombinatorial library had prepared by combining the two libraries at aratio of 1:1. Thus, the comparison of the numbers of phage particlesobtained by screening demonstrated that the phage library prepared usingKL200 is advantageous.

5-2 Comparison of the Activities of Phage Antibodies Obtained byScreening

Then, the selected antibodies were compared to one another with respectto the activity. Clones confirmed to have partial neutralizing activityin the test for the neutralizing activity described in Example 4-11 arethe following four clones:

TETM1 (AIMS3)

TETM13 (AIMS1+AIMS2)

TETM26 (AIMS1+AIMS2)

TETM96 (AIMS1+AIMS2)

Specifically, 3 clones were selected from the library of AIMS1+AIMS2; 1clone was selected from the library of AIMS3. This ratio is comparableto (29:7) which was the ratio of the number of clones obtained byscreening. In addition, the three clones selected from the libraryderived form AIMS1+AIMS2 prepared according to the present inventioncontained TETM13 which had the strongest neutralizing activity. Thus, itwas confirmed that screening of phage library prepared using theselected light chain library KL200 can select superior antibodyefficiently from the aspect of the function.

Example 6 Selection of Phages Binding to a Specific Antigen from a PhageLibrary (Part 2)

The antibody library of the present invention was screened usingdiphtheria toxoid as an antigen. The library used in the screening wasthe same AIMS4 phage library as used in Example 4. This librarycomprised a mixed solution of AIMS3 (a combinatorial library preparedfrom the light chain gene library without selection and the heavy chaingene library) and AIMS1+AIMS2 (a combinatorial library prepared from thelight chain gene library with selection and the heavy chain genelibrary).

6-1 Preparation of Test Tubes to be Used in the Screening

The concentration of the antigen (diphtheria toxoid) was adjusted to 20μg/ml with PBS. A 3-ml aliquot of the antigen solution was added to eachof three test tubes (Maxisorp; Nunc) and incubated at 4° C. for 18 hoursto immobilize the antigen on the inner surface of the tubes. Afterantigen immobilization, the antigen solution was discarded, and 3 ml ofPBS containing 2% skimmed milk was added to each tube. The tubes wereincubated at 25° C. for one hour for blocking.

6-2 Screening Method

A 3-ml aliquot of the same phage library as used in Example 4 (1×10¹⁴CFU) in 9 ml of BBS containing 2% skimmed milk and 0.1% Tween-20 wasadded to each of the three antigen-immobilized test tubes. The tubeswere incubated at 25° C. for 2 hours, and then washed 4 times with PBScontaining 0.1% Tween-20, 4 times with PBS and once sterilized extrapure water (prepared by MilliQ).

Then, phage particles bound to the antigen-immobilized test tubes wererecovered by the procedure as described below. Specifically, 3 ml of 0.1M triethylamine (pH 12.3) was added to each test tube and the tubes wereincubated while being rotated with a rotator at room temperature for 20minutes to dissociate the phage particles from tube surface. Then, 1.1ml of 1 M Tris-HCl buffer (pH6.8) was added to the tubes to neutralizethe solution. The neutralized phage solutions were collected.

6-3 Amplification of the Collected Phages

The collected phage solutions were treated by the same procedure as inExample 4-3 and 4-4 as follows:

(a) the phage was infected to E. coli cells;

(b) helper phage was infected to E. coli cells; and

(c) the resulting phage particles were collected.

The washing was carried out by the same procedure as used in thescreening in Example 4-4.

6-4 Evaluation of phage screening method The nucleotide sequences of thelight chains from clones isolated in this experiment, which wereconfirmed to be expressed, were determined to reveal the originsthereof. As the result, 20 clones were derived from (AIMS1+AIMS2); 16clones were selected from (AIMS4).

Further, it was examined whether these clones contained clones ofpractical utility having high activity. The neutralizing activity ofantibody against diphtheria toxin was determined quantitatively by theCell Culture Method (CCM) using culture cells. The CCM method waspracticed as follows.

The standard antitoxin solution used was prepared by dissolving an ampleof domestic standard diphtheria antitoxin (Lot. 9) in PBS containing 0.2w/v % gelatine, and adjusting the concentration of the antitoxin to 10IU/ml with 3% cs culture medium (containing 1,000 ml of purified watercontaining 9.4 g of Eagle. MEM, 0.3 g of L-glutamine, 200,000 units/2 mlof penicillin, 0.1 g (titer)/0.5 ml of streptomycin, 3.0 g of glucose,30 ml of fetal calf serum, 3 ml of 1% phenol red, and 20 ml of 7 w/v %sodium bicarbonate).

The solution of toxin being tested was prepared by dissolving a vial(Lf/vial=2.5; CD₅₀/vial=1.6×10⁵) of diphtheria toxin (Lot. M59) in PBScontaining 0.2 w/v % gelatine. The concentration of the toxin wasadjusted to 1.6×10⁴ CD₅₀/ml with 3% cs culture medium. The toxinsolution was used after being further diluted with 3% cs culture medium.The cell used was VERO cell, which was passaged in 7% cs culture medium(1,000 ml of purified water containing 9.4 g of Eagle MEM, 0.3 g ofL-glutamine, 200,000 units/2 ml of penicillin, 0.1 g (titer)/0.5 ml ofstreptomycin, 70 ml of fetal calf serum, 3 ml of 1% phenol red, and 20ml of 7 w/v % sodium bicarbonate).

Serial dilutions of (25 μl each) of test antibody and standard antitoxinwere prepared with 3% cs culture medium in a 96-well microtiter plate,and then the diphtheria toxin (25 μl) prepared as described above wasadded thereto. The plated was incubated at 37° C. for 30 minutes, and 50μl of suspension of VERO cell, where the cell density had been adjustedto 3×10⁵ cells/ml with 3% cs culture medium, and 100 μl of 3% cs culturemedium were added thereto. After being sealed, the plate was incubatedin an incubator at 37° C. for 4 days. The endpoint for the standardantitoxin in this assay system was determined based on the changed colorof pH indicator (reference color was orange). The antitoxin titer of atest antibody was defined as a value obtained by multiplying theendpoint antitoxin titer by the dilution fold corresponding to theendpoint of the test antibody. The endpoint of standard antitoxin inthis assay system was determined to be 0.0046 IU/ml.

According to the result, one of the 43 clones tested by CCM method hadthe activity (0.0520 IU/ml) of neutralizing diphtheria toxoid (toxin).On the other hand, no clones having activity was isolated from AIMS3(the light chain gene library prepared without selection). Thesefindings clearly indicate that the antibody library AIMS1+AIMS2 preparedaccording to the present invention was a high-performance libraryexcellently mimicking the in vivo antibody diversity.

The present inventors also isolated a phage clone (DTD10) allowing theproduction of an antibody binding to and neutralizing diphtheria toxin.The nucleotide sequences of the heavy chain and light chain of theabove-mentioned phage clone, and the encoded amino acid sequences areshown under the following SEQ ID NOs:

The nucleotide sequence of heavy chain of DTD10/SEQ ID NO: 61; the aminoacid sequence/SEQ ID NO: 79

The nucleotide sequence of light chain of DTD10/SEQ ID NO: 62; the aminoacid sequence/SEQ ID NO: 80.

Example 7 Selection of Phages Binding to a Specific Antigen from a PhageLibrary (Part 3)

The antibody library of the present invention was screened usingantigens derived from influenza virus. Names of influenza virus strainsare abbreviated hereinafter as follows (virus subtypes are shown inparentheses).

Virus strain name Abbreviation A/New Caledonia/20/99 strain (H1N1) NewCaledonia strain (H1N1) A/Sydney/5/97 strain (H3N2) Sydney strain (H3N2)A/Okuda/57 strain (H2N2) Okuda strain (H2N2) A/Beijing/262/95 strain(H1N1) Beijing strain (H1N1)7-1 Purification of Influenza Virus-Derived Antigen

An antigen derived from influenza virus was purified by a method knownto those skilled in the art as follows. A vaccine strain purchased fromthe National Institute of Infectious Diseases was inoculated toembryonated hen eggs. The eggs were incubated at 33 to 35° C. for 2days, and then allowed to stand still in a cold room at 4° C. overnight.Thus infected chorioallantoic fluid was collected according to aconventional method, and then concentrated by ultra-filtration oranother commonly used biochemical method. The virus sample was purifiedby sucrose density gradient centrifugation. Specifically, theconcentrated virus particles were ultra-centrifuged with a sucrosedensity gradient of 0 to 60% at 35,000 rpm. The fraction at a sucrosedensity of about 40% was collected. The fraction containing virusparticles was treated with ether (ether-inactivated virus). Then,formalin was added to the fraction. The sample was further purified bysucrose density gradient centrifugation to obtain a purified antigen ofinfluenza virus.

7-2 Isolation of Phage Antibodies Using Beijing Strain (H1N1)

7-2-1 Preparation of Test Tubes to be Used in the Screening

The antigen of influenza virus extracted from Beijing strain (H1N1) bythe procedure as described in Example 7-1 was dissolved in PBS at afinal concentration of 12.5 μg/ml. A 4.5-ml aliquot of the antigensolution was added to an immuno-tube (Polysorp), and while beinginverted gently the tube was incubated at 4° C. for 18 hours or at 25°C. for two hours to immobilize the influenza virus antigen on the innersurface of immuno-tube. After the solution of influenza virus antigenwas discarded, 4.5 ml of PBS containing 2% skimmed milk was added to thetube. To avoid non-specific reactions, blocking reaction was carried outby incubating the tube at 4° C. for 18 hours or at 25° C. for one hour.

7-2-2 Screening Method

4.5 ml of a suspension of the AIMS4 phage library, which was the samelibrary as used in Example 4, in PBS containing 2% skimmed milk wasadded to the immuno-tube, on which the antigen had been immobilized,prepared by the procedure as described above. While being inverted thetube was incubated at 25° C. for 2 hours to contact the AIMS4 phagelibrary with the influenza virus antigen immobilized in the innersurface of the tube.

After being contacted as described above, the phage solution wasdiscarded and the tube was washed to remove unbound phage particles fromthe tube. The washing buffer used and the number of washing repetitionsare indicated in Table 8.

TABLE 8 Washing solution Distilled Screening PBS + T PBS water 1st 4 4 12nd 6 6 1 3rd 13 13 1 PBS + T: PBS + 0.1% Tween 20

Then, 4.5 ml of 0.1M triethylamine (pH 12.3) was added to the tube, andwhile being inverted the tube was incubated at room temperature for 20minutes to dissociate the phage particles from the tube. The solutioncontaining the dissociated phage particles was transferred into a freshtube, and immediately 1.1 ml of 1M Tris-HCl (pH 6.8) was added theretoto neutralize the solution.

7-2-3 Amplification of Recovered Phage

E. coli DH12S cells had precultured until OD at 600 nm reach 0.5. Thephage liquid prepared in the above-described step was added to 50 ml (30ml in the secondary or subsequent screening) of the suspension of E.coli DH12S cells (in 2×YT medium). The mixture was incubated at 37° C.for one hour while being shaken to infect the phages havingantigen-binding activity to the E. coli cells.

The above-mentioned E. coli solution was diluted as described below; thefinal volume was 500 ml. (A 3-liter flask was used.)

E. coli solution 61.25 ml 2x YT   425 ml 40% glucose  12.5 ml (finalconcentration: 1%) 100 mg/ml ampicillin  0.5 ml (final concentration:100 μg/ml)

The above-mentioned E. coli diluted solution was further incubated at37° C. for 1 to 2 hours, and the flask was allowed to stand at 4° C.overnight.

The flask stored overnight was again incubated at 37° C. while beingshaken. When OD at 600 nm reached 0.5, the culture solution wastransferred into sterilized centrifugal tubes. Then, the tubes werecentrifuged at 5,000 rpm for 10 minutes at 4° C. to collect thebacterial cells.

The collected bacterial cells were suspended in 1 to 2 ml of 2×YTmedium. An aliquot of the suspension was stored as a glycerol stock(final concentration of glycerol was 20%).

The remaining bacterial cells were suspended in 300 ml (150 ml and 50 mlin the 3rd and 4th screenings, respectively) of 2×YT medium containing100 μg/ml ampicillin, and then cultured. When OD at 600 nm reached 0.5,the helper phage M13K07 solution, which corresponded to 1/10 volume ofthe culture solution, was added to the culture, and incubation wascontinued at 37° C. for one hour while being shaken to infect the helperphage.

An aliquot of the above-mentioned culture solution was plated on an agarplate containing 2×YT medium to estimate the number of helperphage-infected E. coli cells. A fresh culture medium (900 ml of 2×YTmedium containing 100 μg/ml ampicillin and 70 μg/ml kanamycine, whichwas pre-warmed at 37° C.) was added to the remaining culture. Themixture was incubated while being shaken at 37° C. overnight, and thenhelper phage-infected E. coli cells were selected.

After overnight culture, the culture was centrifuged at 7,000 rpm for 10minutes at 4° C. (using sterilized 1,000-ml centrifugal tubes) toprecipitate the bacterial cells, and the resulting supernatant wascollected. A 20% polyethylene glycol solution containing 2.5 M NaCl,which corresponded to ⅕ volume of the supernatant, was added to thesupernatant. The mixture was allowed to stand still at room temperaturefor 20 minutes, and then centrifuged at 8,000 rpm for 15 minutes at 4°C. to precipitate and collect the phage particles.

The supernatant was discarded carefully not to disturb the pellet, andthen the pellet was suspended in sterilized PBS that corresponded to1/10 volume of the culture solution. Then, 20% polyethylene glycolsolution containing 2.5M NaCl, which corresponded to ⅕ volume of PBS,was added to the suspension. The resulting mixture was centrifuged at10,000 rpm for 20 minutes at 4° C. (using sterilized 200-ml centrifugaltubes) to precipitate phages particles. Thus, impurities were removedfrom the phage sample. PBS containing 0.05% NaN₃ which corresponded to1/100 volume of the culture solution was added to the pellet, and thephage particles were suspended by shaking for about two hours using ashaker.

The phage sample prepared in the above-mentioned step was furtherscreened twice. However, the screening yielded only antibodies reactiveto NP protein of influenza virus. These antibodies had no activity ofneutralizing influenza virus.

7-3 Isolation of Subtype-Specific Phage Antibodies Using New CaledoniaStrain (H1N1)

The epitopes which influenza-neutralizing antibodies recognize arebelieved to be present in HA protein on the surface of virus particles.HA protein has many sequence variations among virus strains. Thus, virusstrain-specific antibodies were isolated to obtain neutralizingantibodies.

7-3-1 Preparation of an Antibody Library from which Anti-NP Antibodieshave been Removed by Absorption

Ether-treated inactivated virus particles were prepared using Okudastrain (H2N2) of influenza virus according to the procedure as describedin Example 7-1. The inactivated virus particles were suspended in PBS ata concentration of 20 μg/ml, and 3-ml aliquots of the suspension wereadded to each of three reaction tubes. The tubes were incubated at 4° C.for 18 hours. The suspension was discarded and PBS containing 2% skimmedmilk was added to the tubes. The tubes were incubated for one hour.Then, PBS containing AIMS4 library LOT 000614 and 2% skimmed milk wasadded to the three tubes; the titer of the library used was 1×10¹⁴ CFU/9ml. The tubes were incubated at room temperature (25° C.) for two hourswhile being agitated. The library after incubation was used as an AIMS4antibody library from which anti-NP antibodies had been removed byabsorption.

7-3-2 Screening with Antigens of Interest Derived from New CaledoniaStrain (H1N1)

Formalin-treated inactivated virus particles of New Caledonia strain(H1N1) were prepared by the same procedure as described above, andsuspended in PBS at a concentration of 100 μg/ml. 3-ml aliquots of thesuspension were added to each of three reaction tubes. The tubes wereincubated at 4° C. for 18 hours. The suspension was discarded and PBScontaining 2% skimmed milk was added to the tubes to preventnon-specific reaction. The tubes were incubated for one hour. Then, theAIMS4 library, from which anti-NP antibodies had been removed byabsorption, prepared in Example 7-3-2 was added to the three tubes. Thetubes were incubated at 25° C. for two hours while being agitated. Afterincubation, the tubes were washed with PBS; the number of washingrepetitions is shown below in Table 9.

After washing, 3 ml of 0.1M triethylamine (pH 12.3) was added to eachtube. The tubes were incubated at room temperature for 20 minutes whilebeing agitated with a rotator to dissociate phage particles from theantigen. 1 ml of 1M Tris-HCl (pH6.8) was added to the tubes toneutralize the liquid (recovery of phage particles bound to theantigen). E. coli DH12S cells were precultured in 50 ml of 2×YT mediumuntil OD at 600 nm reached 0.5 to 1.0. Then, the antigen-bound phageparticles were incubated with the E. coli cells at 37° C. for one hourwhile being shaken to infect the phage to the bacteria.

The recovered phage particles were amplified by the same procedure asdescribed in Example 7-2-3. After amplification, anti-NP antibodies wereremoved by absorption according to the same procedure as described inExample 7-3-1. The titer of the phage solution was determined asdescribed below. Then, screening was carried out in the same way byusing the solution of amplified phage particles. The screening step wasrepeated three times.

7-3-3 Determination of Phage Titer

The titer of phage solution to be used in the next round of screeningwas previously determined by the procedure as described below.

A serial dilution was prepared as follows:

[1] 1×10⁻² dilution: 10 μl of phage solution+990 μl of PBS

[2] 1×10⁻⁴ dilution: 10 μl of the dilute in [1]+PBS 990 μl

[3] 1×10⁻⁶ dilution: 10 μl of the dilute in [2]+990 μl of PBS

[4] 1×10⁻⁸ dilution: 10 μl of the dilute in [3]+990 μl of PBS

[5] 1×10⁻¹⁰ dilution: 10 μl of the dilute in [4]+990 μl of PBS

[6] 1×10⁻¹² dilution: 10 μl of the dilute in [5]+990 μl of PBS

990 μl of DH12S was combined with each of 10-μl aliquots of the dilutesprepared in [4], [5], and [6] in the serial dilution. The mixtures wereincubated at 37° C. for one hour for infection, and 100-μl aliquotsthereof were plated on LBGA plates, followed by incubation at 30° C. for18 to 24 hours. The resulting colonies grown were counted.

When the number of colonies grown was 50 colonies/plate or more, suchplates were selected from those prepared in [4], [5], and [6]; thecolony counts in such plates were adopted as data.

A formula for determining the phage titer for the next-round screening(in the case described in [4])Phage titer=(the total number of colonies/plate)×(1×10⁸)×10³ phages/ml

TABLE 9 New Caledonia strain (H1N1) after being absorbed with Okudastrain (H2N2) The number of washing Screening repetitions Input*Output** 1/(Input/Output) 1st 4 1.0 × 10¹⁴ 9.1 × 10⁸ 1/(1.1 × 10⁶) 2nd 84.6 × 10¹³ 1.1 × 10⁸ 1/(5.1 × 10⁶) 3rd 16 4.0 × 10¹³ 4.7 × 10¹⁰ 1/(8.5 ×10²) *From the result obtained in Example 7-3-3; unit: (cfu/ml).**Calculated from the number of phage particles prior to amplification,which were recovered in the screening step in Example 8-3-2; unit:(cfu/ml).7-4 Method for Isolating Neutralizing-Antibodies Specific to the H3N2Subtype7-4-1 Screening with Antigens of Interest Derived from Sydney Strain(H3N2)

Formalin-Treated Inactivated Influenza Virus Particles of Sydney strain(H3N2) were suspended in PBS at a concentration of 100 μg/ml, and thesuspension (4.5 ml per tube) was added to three Nunc Polysorp tubes. Thetubes were incubated at 4° C. for 18 hours. The suspension was discardedand PBS containing 2% skimmed milk was added to the tubes. The tubeswere incubated for one hour for blocking. Then, the AIMS4 library, fromwhich anti-NP antibodies had been removed by absorption, prepared inExample 7-3-1 was added to the three tubes. The tubes were incubated atroom temperature (25° C.) for two hours while being agitated using arotator.

After incubation, the tubes were washed with PBS; the number of washingrepetitions is shown below in Table 10.

After washing, 3 ml of 0.1M triethylamine (pH 12.3) was added to eachtube. The tubes were incubated at room temperature for 20 minutes whilebeing agitated with a rotator to dissociate phage particles from theantigen. 1 ml of 1 M Tris-HCl (pH6.8) was added to the tubes toneutralize the solution (recovery of phage particles bound to theantigen).

E. coli DH12S cells were pre-cultured in 50 ml of in 2×YT medium untilOD at 600 nm reached 0.5 to 1.0. Then, the antigen-bound phage particleswere incubated with the E. coli cells at 37° C. for one hour while beingshaken to infect the phage to the bacterial cells.

The recovered phage particles were amplified by the same procedure asdescribed in Example 7-2-3. After amplification, anti-NP antibodies wereremoved by absorption according to the same procedure as described inExample 7-3-1. After the titer of the phage solution was determined bythe same method as described in 7-3-3 (Table 11), then screening wascarried out in the same way by using the solution of amplified phageparticles. The screening step was repeated three times.

TABLE 10 Sydney strain (H3N2) after being absorbed with Okuda strain(H2N2) The number of washing Screening repetitions Input* Output*1/(Input/Output) 1st 4 1.0 × 10¹⁴ 1.0 × 10⁹ 1/(1.0 × 10⁵) 2nd 8 4.0 ×10¹³ 3.3 × 10⁷ 1/(1.2 × 10⁶) 3rd 16 5.6 × 10¹³ 2.8 × 1010 1/(2.6 × 10³)*From the result obtained in Example 7-3-3; unit: (cfu/ml). **Calculatedfrom the number of phage particles prior to amplification, which wererecovered in the screening step in Example 7-3-2; unit: (cfu/ml).7-5 Evaluation of Phage Antibodies Neutralizing Influenza Virus7-5-1 Induction of the Expression of Fab-Type Antibody

The present inventors have found that when phage-infected E. coli iscultured for a long period after induction of the expression, not phagebut the Fab-type antibody is secreted from E. coli into the culturesupernatant.

E. coli cells from the colonies of phage-containing E. coli obtainedfrom the steps in Example 7-2-3, 7-3-3, and 7-4-1 were inoculated to2×YT medium containing 0.1% glucose and 100 μg/ml ampicillin. Thebacterial cells were cultured at 30° C. When OD at 600 nm reached about0.5, IPTG was added to the culture at a final concentration of 1 mM.Then, culture was continued at 30° C. for 18 hours to induce theexpression of phage antibodies.

A glycerol stock of E. coli was prepared by adding glycerol at a finalconcentration of 30% to another culture prepared by incubating for 18hours without adding IPTG.

7-5-2 Confirmation of Characteristics of Fab-Type Antibody by ELISA

1.5 ml of the culture solution, in which the Fab-type antibody had beenexpressed, prepared in Example 7-5-1 was placed in a tube, andcentrifuged at 10,000 rpm for 5 minutes at 4° C. The resulting culturesupernatant was collected, and NaN₃ was added thereto at a finalconcentration of 0.1%. The supernatant was used as an ELISA sample.

Then, to each well of a 96-well ELISA plate, 100 μl of the antigen wasadded at the same concentration as used in the screening. The plate wasincubated at 4° C. for 18 hours to immobilize the antigen on the surfaceof each well of the plate. Then, 200 μl of PBS containing 2.5% BSA wasadded to each well, and the plate was incubated at 4° C. for 18 hoursfor blocking.

Before the ELISA test was conducted, the blocking solution was discardedand the plate was washed once with PBS. 100 μl of the supernatant of theculture where the induction was carried out in Example 7-5-1 was addedto each well. After the plate was incubated at 25° C. for one hour, thewells were washed 4 times with PBS. After A POD (peroxidase)-conjugatedanti-human IgG (MBL; Cat. #206 LOT150) was diluted 5,000 times, a 100-μlaliquot of the solution was added to each well. The plate was incubatedat 25° C. for one hour (or at 37° C. for one hour, while being shaken atroom temperature at 250 rpm for 30 minutes). Then, the plate was washedfour times with PBS, and 100 μl of a substrate solution was added toeach well. The substrate solution used was prepared as follows: 12 ml ofH₂O₂ was added at a final concentration of 0.01% to 0.1 M citricacid-disodium phosphate (pH 5.1), and then a tablet of OPD (Wako,Biochemical Grade, Code #158-01671) was added thereto. After 15 to 30minutes, the absorbance (OD at 492 nm) of each well was measured with aplate reader.

According to the result obtained by ELISA, it was revealed thatneutralizing antibodies exhibiting strain-specific reactivity were 1clone specific to New Caledonia strain (H1N1) and 3 clones specific toSydney strain (H3N2). The reactivities of these four clones areindicated in Table 12.

TABLE 11 New Caledonia Sydney strain strain (H3N2) after being (H1N1)after being absorbed with absorbed with Okuda Okuda strain Antigenstrain (H2N2) (H2N2) The number of 48 48 tested colonies Having no gene37 29 deletion * ELISA positive 35 27 Specific to strain- 1 2 dependentantigen * Having no sequence deletion, according to the result ofnucleotide sequencing of gene

TABLE 12 Antigen-immobilized plate Antigen New Caledonia Okuda Sydneyused in strain strain strain screening Clone No. (H1N1) (H2N2) (H3N2)New Caledonia NC1 1.991 0.052 0.098 strain (H1N1) Sydney strain SY390.090 0.080 1.612 (H3N2) Sydney strain SY47 0.080 0.056 2.859 (H3N2)Control IF8 2.966 2.438 2.923 Anti-NP Negative control 0.090 0.057 0.0657-5-3 Plasmid Purification and Sequencing

E. coli cells were cultured in 2×YT medium (containing 0.1% glucose and200 μg/ml ampicillin) overnight, and plasmids were purified from thecells using a DNA extractor (PI-50; Kurabo) according to the supplier'sinstruction. The purified plasmids were treated with Rnase, which wasadded at a final concentration of 20 μg/ml, and then treated withphenol-chloroform. The plasmids were stored at −20° C. or lowertemperature.

The sequences of antibody genes corresponding to the obtained phageantibodies were determined by sequencing. Sequencing was carried out bythe dideoxy method using a DNA sequencer (Aloka; L1-COR4200L (S)-2); thereagent used was thermo sequence kit (US78500; Amersham Pharmacia).

The sequencing primers used for the heavy chain and light chain were asfollows:

(SEQ ID NO: 97) heavy chain: T7 Promoter IDR700 dye-labeled primer 50 μ1 5′TAATACgACTCACTATAggg3′ (20 mer)

(SEQ ID NO: 98) light chain: custom primer 3′huCH1J    IDR800 dye-labeled primer 50 μl 5′ATTAATAAgAgCTATCCCgg3′ (20 mer)

The number of clones having no sequence deletion, which was determinedby nucleotide sequencing for the genes, is indicated in Table 11. Genescorresponding to strain-specific clones and amino acid sequences areshown in Sequence Listing.

7-5-4 Neutralization Test for Influenza Virus

A suspension of MDCK cells was aliquoted into each well of a 96-wellflat-bottomed plate (Corning; cat. #3596) (approximately 10⁴cells/well). The plate was incubated in a CO₂ incubator at 37° C. sothat the bottom surface of each well could be covered with a monolayersheet of cell. On the next day, the respective antibodies to be tested,after being diluted 4 times with MEM culture medium containing 0.2% BSA(fraction V; Sigma Chemical Co.) were added to wells of a 96-wellround-bottomed plate. 25 μl of an influenza virus solution (4×10⁴FFU/ml) diluted with MEM culture medium containing 0.2% BSA was combinedwith 25 μl of the diluted antibody solution or control solution. Themixture was incubated at 37° C. for 60 minutes.

25 μl each of the mixed solutions was added to the MDCK cells in wellsof the above-mentioned 96-well plate. The virus particles were allowedto adhere to the cells by incubating the plate at 37° C. for 60 minutes.

After this treatment, each well was washed with PBS. 100 μl of MEMculture medium containing 0.5% tragacanth gum and 5 μg/ml trypsin wasadded to each well, and then culture was continued at 37° C. for 24hours.

After cultivation, each well was washed with PBS. 100% ethanol was addedto each well, and then the plate was incubated at room temperature for10 minutes. The wells were dried using a hair dryer. After PAP staining,the number of focuses was determined.

The neutralizing activity of an antibody was defined as a decrement ofthe number of focuses relative to that without adding any antibody(positive control).

As seen below in Table 13, the Fab-type antibodies corresponding toClone Nos: NC1, SY39, and SY47 exhibited significantly high activity ofneutralizing influenza virus.

TABLE 13 New Caledonia strain (H1N1) Sydney strain (H3N2) ReductionReduction Clone No. Virus titer of focus Virus titer of focus (Dilutionfold) (FFU/well) count (%) (FFU/well) count (%) NC1 (1:1) 26 71.3 NDSY39 (1:1) ND 3 94.9 SY47 (1:1) ND 12.5 78.6 IF8 (1:1) 71 21.5 66 0*AS1296 (1:100) 43.5 51.9 1 98.3 *C179 (1:100) 12.5 98.3 ND *a-AichH3(1:100) ND 2.5 95.7 Positive control 90.5 0 58.5 0 FFU: Focus FormingUnit ND: Not Done *Antibody known to have neutralizing activity${{Reduction}\mspace{14mu}{of}\mspace{14mu}{focus}\mspace{14mu}{count}\mspace{14mu}(\%)} = {\frac{\left( {{Titer}\mspace{14mu}{of}\mspace{14mu}{positive}\mspace{14mu}{control}} \right) - \left( {{Titer}\mspace{14mu}{of}\mspace{14mu}{sample}\mspace{14mu}{virus}} \right)}{\left( {{Titer}\mspace{14mu}{of}\mspace{14mu}{positive}\mspace{14mu}{control}} \right)} \times 100}$(Titer: FFU/well)7-5-5 Conclusions Concerning the Evaluation

As predicted, the antibodies of virus strain-specific phage antibodyclones exhibited high activity of neutralizing influenza virus. Namely,the clones are: clone NCl specific to New Caledonia strain (H1N1);clones SY39 and SY47 specific to Sydney strain (H3N2). The nucleotidesequences of the heavy chain and light chain in these phage clones, andthe amino acid sequences encoded by the same are shown below under thefollowing SEQ ID NOs:

Clone No. Heavy chain Light chain NC1 (nucleotide sequence) 67 68 NC1(amino acid sequence) 85 86 SY39 (nucleotide sequence) 69 70 SY39 (aminoacid sequence) 87 88 SY47 (nucleotide sequence) 71 72 SY47 (amino acidsequence) 89 90

Because the antibody genes are already available and characterized, whenintending to use practically these types of antibody variable regionsfor clinical purposes, one can construct complete immunoglobulinmolecules from the antibody variable regions or conjugate the regionswith an enzyme by a relatively simple method. Furthermore, the activitycan be improved by introducing artificial mutations.

Example 8 Selection of Phages Binding to a Specific Antigen from a PhageLibrary (Part 4)

The antibody library of the present invention was screened usingantigens derived from varicella-zoster virus (VZV).

8-1 Isolation of Phage Antibodies Reactive to VZV

8-1-1 Preparation of Test Tubes to be Used in the Screening

VZV-infected human embryonic lung cell (HEL) was available from somepublic organizations. The cells in PBS were lysed by sonication. Theproteins gE and gH were prepared from the lysate according to the methodof Shiraki et al. (Shiraki, K., Takahashi, M. J. Gen. Virol. 1982, 61:271-5). A solution of gH protein whose concentration was adjusted to 25μg/ml was used as a VZV antigen solution. A 4.5-ml aliquot of the VZVantigen solution was added to an immuno-tube (Maxisorp), and while beinginverted gently the tube was incubated at 4° C. for 18 hours or at 25°C. for two hours to immobilize the VZV antigen on the inner surface ofimmuno-tube. After the VZV antigen solution was discarded, 4.5 ml of PBScontaining 2% skimmed milk was added to the tube. To avoid non-specificreactions, blocking reaction was carried out by incubating the tube at4° C. for 18 hours or at 25° C. for one hour.

8-1-2 Screening Method

4.5 ml of a suspension of the AIMS4 phage library in PBS containing 2%skimmed milk was added to the immuno-tube, on which the antigen had beenimmobilized, prepared by the procedure as described above. While beinginverted the tube was incubated at 25° C. for 2 hours to contact theAIMS4 phage library with the VZV antigen immobilized in the innersurface of the tube.

After being contacted as described above, the phage solution wasdiscarded and the tube was washed to remove unbound phage particles fromthe tube. The washing buffer used and the number of washing repetitionsare indicated in Table 14. The term “washing” refers to a series oftreatments comprising adding a buffer, inverting the tube containing thebuffer, and discarding the buffer.

TABLE 14 Washing Input Output In/Out 1^(st) 2% skim − PBS 4 1.3 × 10¹⁴1.4 × 10⁹ 9.6 × 10⁴ times + PBS once 2^(nd) 2% skim − PBS 5 3.1 × 10¹³ 9.3 × 10⁴ 3.3 × 10⁸ times + PBS 15 times 3^(rd) 2% skim − PBS 5 4.6 ×10¹³ 3.6 × 10⁶ 1.3 × 10⁷ times + PBS 20 times 4^(th) 2% skim − PBS 5 5.0× 10¹³ 2.5 × 10⁸ 2.0 × 10⁵ times + PBS 20 times

Then, 4.5 ml of 0.1M triethylamine (pH 12.3) was added to the tube, andwhile being inverted the tube was incubated at room temperature for 20minutes to dissociate the phage particles from the tube. The solutioncontaining the dissociated phage particles was transferred into a freshtube, and immediately 1.1 ml of 1 M Tris-HCl (pH 6.8) was added theretoto neutralize the solution.

Amplification of recovered phage particles was achieved by the sameprocedure as described in Example 7-2-3, and then the titer of the phagesolution after amplification was determined by the same method asdescribed in Example 7-3-3.

The screening step as described above was repeated three times.

8-2 The Step of Assessing the Antigen-Binding Activity Using CultureSupernatants of E. coli Cells by ELISA

The antigen-binding activity was assessed by the same ELISA method asused in Example 7-5-2. The VZV antigen was dissolved in PBS to aconcentration of 12.5 μg/ml. A 100-μl aliquot of the solution was addedto each well of a 96-well plate, and the plate was incubated at 4° C.for 18 hours to immobilize the antigen on the surface of each well of96-well plate. Then, 200 μl of 2.5% BSA was added to each well and theplate was incubated at 4° C. for 18 hours for blocking.

After blocking, the blocking solution of the 96-well plate wasdiscarded. The plate was washed once with PBS, and then 100 μl ofculture supernatant of phage-infected E. coli cells prepared by the sameprocedure as in Example 8-5-2 was added to each well. The plated wasincubated at 25° C. for one hour. The subsequent steps were conducted inthe same way as in Example 8-5-2.

As seen in the Table indicated above, the recovery rate (output) wasincreased in the screening for the third time, and thus 12 clones weretested for the activity by ELISA. According to the result, 1 cloneexhibited strong activity. Then, the fourth screening was carried outand 48 clones were tested by ELISA. The clones were all positive whilethe activities were weak.

8-3 Comparison of the Reactivity Between gE and gH, and Sequencing

Antibodies against sugar chains were predicted to be reactive to othermoieties of virus particle, and to have low specificity and lowneutralizing activity. Thus, ELISA was also carried out for antibodiesagainst gE abundantly containing glycoprotein. Thus, antibodies havingsignificant reactivity to gE were excluded based on the result obtained.

Based on this criterion, 29 clones having significant reactivity to gHwere selected (Table 15), and the nucleotide sequences of the cloneswere analyzed (sequenced) by the same procedure as described in Example7-5-3. Sequencing revealed 16 types of nucleotide sequences. The culturesupernatants of representative clones were collected and concentratedwith saturated ammonium sulfate. After dialysis, the samples were testedfor the neutralizing activity.

TABLE 15 Comparison of binding Type of activity between gH sequenceClone name and gE by ELISA 1 VZ9 gH ≦ gE low 2 VZ10, VZ34, VZ75 gH > gElow 3 VZ11 gH >> gE 4 VZ13, VZ15, VZ19 gH < gE 5 VZ24 gH > gE low 6 VZ36gH ≦ gE high 7 VZ49, VZ66 gH = gE 8 VZ60, VZ76 gH > gE low 9 VZ64, VZ33,VZ58 gH ≧ gE 10 VZ68 gH < gE 11 VZ77 gH > gE low 12 VZ85 gH < gE 13VZ86, VZ96 gH ≧ gE low 14 VZ89,VZ80,VZ81 gH = gE low 15 VZ94,VZ62 gH >gE low 16 VZ100 gH ≧ gE 17 VZ102 gH > gE low Underline: clone tested forneutralizing activity; Bold italic: clone found to have strongneutralizing activity.8-4 Neutralization Test of Obtained Antibodies for VZV Virus8-4-1 Preparation of Cell-Free Varicella-Zoster Virus (VZV Oka Strain)to be Used in the Test

When approximately 50% of human embryonic lung cells (HEL) beingcultured showed alterations specific to infected cells (CPE: cytopathiceffect), the cells were harvested using PBS containing EDTA (0.04 to0.1%) (without trypsin) and centrifuged at a low speed. The cellsharvested were suspended in SPGC storage (sterilized PBS containing 5%sucrose, 0.1% sodium glutaminate and 10% fetal calf serum).

The cells were lysed by sonication or freeze-and-thaw plus sonication torelease the virus particles. The lysate was centrifuged at 3,000 rpm for10 minutes, and the resulting supernatant was used as a virus solution,which was stored at −85° C. Using an aliquot of the solution, the titer(infectivity) was determined with HEL cells. Specifically, the virussolution was diluted appropriately with SPGC storage, and a 0.2-mlaliquot of the dilute was scattered onto HEL cells that had been placedin a 6-cm dish to inoculate the virus to the cells. The inoculation wasachieved as follows: the virus solution was being scattered repeatedlyback and forth equally onto the HEL cells for one hour so that the cellscould be covered with the virus solution and the cell surface was notdried. After inoculation, culture medium was added to the dish, and thedish was incubated for 4 to 6 days. Then, the culture medium wasremoved. The cells were fixed with 5% formalin according to a standardmethod, and stained with methylene blue; the number of plaques wasdetermined under a stereoscopic microscope. The number of plaques in aunit volume (PFU/ml) was taken as an index (titer) of infectious virus.

8-4-2 Neutralizing Activity Test

The virus solution prepared as described above was diluted to 100PFU/0.1 ml with SPGC storage. A 0.3-ml aliquot of the diluted virussolution was combined with 0.3 ml of an antibody solution that had beendiluted 10 times with SPGC. The mixture was incubated at 37° C. for onehour. A 0.2-ml aliquot of the mixture was scattered onto HEL cells thathad been placed in a 6-cm dish to inoculate the virus to the cells. Theinoculation was achieved as follows: the virus liquid was beingscattered repeatedly back and forth equally onto the HEL cells for onehour so that the cells could be covered with the virus solution and thecell surface was not dried.

After inoculation, 5 ml of culture medium was added to the dish, and thedish was incubated for 4 to 6 days. Then, the culture medium wasremoved. The cells were fixed with 5% formalin according to a standardmethod, and stained with methylene blue; the number of plaques wasdetermined under a stereoscopic microscope.

The neutralizing activity of an antibody toward virus assessed bycomparing the number of plaques formed in the absence of antibody (PBSalone) as a control with that in the presence of antibody (i.e., thenumber of plaques is decreased in the presence of a neutralizingantibody). As seen below in Table 16, the clones of Nos. 10, 24, and 94had strong neutralizing activity.

TABLE 16 The number of plaques in Antibody neutralization testNeutralizing clone name 1st test 2nd test 3rd 4th activity Control 4246, 48, 50, (PBS) VZ9 * VZ10 0 0 +++ VZ11 30 25 +− VZ13 47 48 − VZ24 0 0+++ VZ36 14 15 + VZ49 40 43 − VZ60 15 9 + VZ64 41 42 − VZ77 36 * VZ86 4650 − VZ89 32 33 +− VZ94 1 2 ++ VZ100 29 28 +− VZ102 19 24 +−Neutralizing activity: Very strong, +++; Strong, ++; Detectable, +;Unassignable, +−; None, − *: incapable measurement because ofcontamination of microorganism.8-4-3 Conclusions Concerning the Neutralization Test

According to the present invention, three phage clones were obtained,which strongly suppressed plaque formation, i.e., clones VZ10, VZ24, andVZ94. The nucleotide sequences of the heavy chain and light chain in thephage clones, and the amino acid sequences encoded by the same are shownbelow under the following SEQ ID NOs:

Clone. No. Heavy chain Light chain VZ10 (nucleotide sequence) 73 74 VZ10(amino acid sequence) 91 92 VZ24 (nucleotide sequence) 75 76 VZ24 (aminoacid sequence) 93 94 VZ94 (nucleotide sequence) 77 78 VZ94 (amino acidsequence) 95 96

The nucleotide sequences of the clones are different from one another.Thus, when used practically for clinical purposes, the neutralizingactivities can be synergistic. Because the antibody genes are alreadyavailable and characterized, when intending to practically use theseantibody variable domains, one can construct complete immunoglobulinmolecules containing the constant domains from the antibody variabledomains or conjugate the domains with an enzyme by a relatively simplemethod.

INDUSTRIAL APPLICABILITY

The present invention provided an antibody library which containsantibody molecules having functionally active conformation at a highrate, or a library of genes encoding the same. Firstly, the preparationof the gene library of the present invention comprises selecting genesencoding the light chain variable region capable of forming functionallyactive conformation in combination with the heavy chain variable domain.The gene library of the present invention constructed by combining theresulting light chain variable region genes selected with the heavychain variable region genes can mimic the antibody repertoire comprisingfunctionally active antibodies. The antibody library of the presentinvention constructed via the step of selecting functional antibodymolecules can be assumed to mimic, in vitro, the in vivo antibodyrepertoire.

An antibody library containing functionally active antibodies at a highrate can be prepared based on the gene library of the present invention.Functionally active antibodies can be isolated efficiently by screeningsuch an antibody library. In preparing antibody libraries known to thoseskilled in the art, the major interest had been in only increasing thenumber of antibody variations in such libraries. However, the presentinventors focused on the quality of antibodies constituting suchlibraries and then established a means of improving the quality in thepresent invention. Thus, the present invention provides an entirelydifferent viewpoint on the in vitro construction of antibody librariesand method for screening antibodies.

The heavy chain diversity can be used to full advantage in antibodylibraries prepared according to the present invention. This advantage isnot confined to allowing to mimic the in vivo antibody diversity invitro. Based on the present invention, for example, a method forintroducing artificial mutations, such as error-prone PCR, can be usedto full advantage. Specifically, in the present invention,theoretically, the heavy chain variations due to the introducedmutations fully contribute to the antibody variations. Because the lightchain which allows the re-holding of a functionally active antibody incombination with the heavy chain, are selected according to the presentinvention. It contrasts with that, in a previous library known to thoseskilled in the art, when the light chain does not allow the formation ofa functionally active antibody molecule, no antibody activity isdetectable in spite of the presence of effective mutation in the heavychain and thus screening may results in failure.

1. A method for preparing a gene library comprising combinations ofdifferent light chain variable region genes and heavy chain variableregion genes of immunoglobulin, the method comprising: (a) selecting aplurality of human derived different light chain variable region genesencoding light chain molecules, wherein the light chain variable regiongenes are selected by detecting a holding structure of a complex throughan association of a heavy chain variable chain molecule with a lightchain variable chain molecule to form Fab, further wherein theassociation is detected by both an antibody of I) and an antibody of II)binding to Fab, when the light chain molecule is combined with anexpression product of a heavy chain variable region gene encoding theheavy chain variable chain molecule, wherein I) is an antibody againstthe light chain, and II) is an antibody against the heavy chain or Fab,and wherein the plurality of different light chain variable region genesis 100 for each of κ chain genes and λ chain genes; (b) recovering eachof the human derived different light chain variable region gene selectedin step (a) from the heavy chain variable region gene; (c) constructinga gene library consisting of a collection of the human derived differentlight chain variable region genes recovered in step b); and (d)combining the gene library of light chain variable region genes obtainedin step (c) with one or more libraries of genes encoding human derivedheavy chain variable regions having a full repertoire of heavy chainvariable region genes, whereby the resulting gene library maintainsdiversity in the heavy chain variable region.
 2. A gene librarycomprising combinations of different light chain variable region genesand heavy chain variable region genes of immunoglobulin, wherein thegene library is obtained by: (a) selecting a plurality of human deriveddifferent light chain variable region genes encoding light chainmolecules, wherein the light chain variable region genes are selected bydetecting a holding structure of a complex through an association of aheavy chain variable chain molecule with a light chain variable chainmolecule to form Fab, further wherein the association is detected byboth an antibody of I) and an antibody of II) binding to Fab, when thelight chain molecule is combined with an expression product of a heavychain variable region gene encoding the heavy chain variable chainmolecule, wherein I) is an antibody against the light chain, and II) isan antibody against the heavy chain or Fab, and wherein the plurality ofdifferent light chain variable region genes is 100 for each of κ chaingenes and λ chain genes; (b) recovering each of the human deriveddifferent light chain variable region gene selected in step (a) from theheavy chain variable region gene; (c) constructing a library consistingof a collection of the human derived different light chain variableregion genes recovered in step b); and (d) combining the library oflight chain variable region genes obtained in step (c) with one or morelibraries of genes encoding human derived heavy chain variable regionshaving a full repertoire of heavy chain variable region genes, wherebythe resulting gene library maintains diversity in the heavy chainvariable region.
 3. An antibody library that comprises antibody proteinsencoded by genes from a gene library, wherein the gene library isobtained by: (a) selecting a plurality of human derived different lightchain variable region genes encoding light chain molecules, wherein thelight chain variable region genes are selected by detecting a holdingstructure of a complex through an association of a heavy chain variablechain molecule with a light chain variable chain molecule to form Fab,further wherein the association is detected by both an antibody of I)and an antibody of II) binding to Fab, when the light chain molecule iscombined with an expression product of a heavy chain variable regiongene encoding the heavy chain variable chain molecule, wherein I) is anantibody against the light chain, and II) is an antibody against theheavy chain or Fab, and wherein the plurality of different light chainvariable region genes is 100 for each of κ chain genes and λ chaingenes; (b) recovering each of the human derived different light chainvariable region gene selected in step (a) from the heavy chain variableregion gene; (c) constructing a library consisting of a collection ofthe human derived different light chain variable region genes recoveredin step b); and (d) combining the library of light chain variable regiongenes obtained in step (c) with one or more libraries of genes encodinghuman derived heavy chain variable regions having a full repertoire ofheavy chain variable region genes, whereby the resulting librarymaintains diversity in the heavy chain variable region.
 4. The method ofclaim 1, wherein the antibody against the light chain for selecting theκ chain genes and λ chain genes is an antibody against the kappa chainand an antibody against the lambda chain, respectively.
 5. The method ofclaim 4, wherein the κ chain genes and λ chain genes are selected withan ELISA for the detection of binding between the antibody of I) and theantibody of II) to Fab, wherein I) is an antibody against the kappachain or an antibody against the lambda chain and II) is an antibodyagainst the heavy chain or Fab.
 6. The method of claim 5, wherein eachof 100 of the κ chain genes and λ chain genes are selected from thosehaving the highest reactivity rank detected by the ELISA.
 7. The methodof claim 1, wherein the library of genes encoding the human derivedheavy chain variable regions is obtained from naive B cells.
 8. Themethod of claim 1, wherein redundancy within the plurality of differentlight chain variable region genes selected is removed prior to step (b).9. The gene library according to claim 2, wherein the antibody againstthe light chain for selecting the κ chain genes and λ chain genes is anantibody against the kappa chain and an antibody against the lambdachain, respectively.
 10. The antibody library according to claim 3,wherein the antibody against the light chain for selecting the κ chaingenes and λ chain genes is an antibody against the kappa chain and anantibody against the lambda chain, respectively.